IAP BIR domain binding compounds

ABSTRACT

Disclosed is an isomer, enantiomer, diastereoisomer or tautomer of a compound represented by Formula I:  
                 
 
or a salt thereof, in which R 1 , R 2 , R 100 , R 200 , A, A 1 , B, B 1 , BG, n, Q and Q 1  are substituents described. Also disclosed is the use of compounds of Formula 1 to treat proliferative disorders.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of pending U.S. patent applicationSer. No. 11/583,816, filed Oct. 20, 2006, which claims priority frompreviously filed U.S. Provisional Patent Application Ser. No.60/729,727, filed Oct. 25, 2005 and U.S. Provisional Patent ApplicationSer. No. 60/830,662, filed Jul. 14, 2006, the entire contents of whichare hereby incorporated by reference.

FIELD OF THE INVENTION

The present invention concerns compounds that bind to IAP BIR domains,and more particularly the BIR2 and BIR3 domains, and are useful to treatproliferative disorders.

BACKGROUND OF THE INVENTION

Apoptosis, or programmed cell death, typically occurs in the developmentand maintenance of healthy tissues in multicellular organisms. Apoptoticpathways are known to play a critical role in embryonic development,viral pathogenesis, cancer, autoimmune disorders, and neurodegenerativediseases, as well as other events. Alterations in an apoptotic responsehas been implicated in the development of cancer, autoimmune diseases,such as systemic lupus erythematosis and multiple sclerosis, and inviral infections, including those associated with herpes virus,poxvirus, and adenovirus.

Caspases, a class of cysteine proteases, are known to initiate apoptosisafter they have been activated. Inhibitors of apoptosis proteins (IAPs)are a family of proteins, which contain one to three baculovirus IAPrepeat (BIR) domains, namely BIR1, BIR2, and BIR3, and may also containa RING zinc finger domain at the C-terminus. Examples of human IAPsinclude, XIAP, HIAP1 (also referred to as cIAP2), and HIAP2 (cIAP1) eachhave three BIR domains, and a carboxy terminal RING zinc finger. NAIPhas three BIR domains (BIR1, BIR2 and BIR3), but no RING domain, whereasLivin and ILP2 have a single BIR domain and a RING domain. The prototypeX chromosome linked inhibitor of apoptosis (XIAP) can not only inhibitsthe activated caspases by direct binding to the caspases, but XIAP canalso remove caspases and the second mitochondrial activator of caspases(Smac) through the ubiquitylation-mediated proteasome pathway via the E3ligase activity of a RING zinc finger domain. The BIR3 domain of XIAPbinds and inhibits caspase-9, which can activate caspase-3. Thelinker-BIR2 domain of XIAP inhibits the activity of effector caspases-3and -7-. The BIR domains have also been associated with the interactionsof IAPs with tumor necrosis factor-associated factor (TRAFs)-1 and -2,and to TAB1.

Overall the IAPs function as a ‘constraint’ to apoptosis and maydirectly contribute to the tumor progression and resistance topharmaceutical intervention. Interestingly, results demonstrate thatresistance to apoptosis can be decrease by siRNA and antisense directedagainst specific IAP's in the cells. Hence, suggesting that interferingwith the activity of the IAP's might prove advantageous in sensitizingdisease cells to apoptosis.

A series of endogenous ligands are capable of interfering withIAP-caspase interactions. The X-ray crystallographic structure of XIAPBIR2 and BIR3 reveal a critical binding pocket and groove on the surfaceof each BIR domain. Two mammalian mitochondrial proteins, namely secondmitochondria-derived activator of caspases (Smac) and Omi/Htra2, andfour Drosophila proteins (Reaper, HID, Grim, and Sickle), whichinterfere with IAP function by binding to these sites on theirrespective BIR domain, have been identified. Each of these IAPinhibitors possesses a short amino-terminal tetrapeptide, AXPY orAVPI-like, sequence that fits into this binding pocket and disruptsprotein/protein interactions such as IAP-caspase interactions. Althoughthe overall folding of individual BIR domains is generally conserved,there are alterations in the amino acid sequences that form the bindingpocket and groove. As such, binding affinities vary between each of theBIR domains.

A number of compounds have been described, which reportedly bind XIAPincluding Wu et al., Chemistry and Biology, Vol. 10, 759-767 (2003);United States published patent application number US2006/0025347A1;United States published patent application number US2005/0197403A1;United States published patent application number US2006/0194741A1. Someof the aforesaid compounds, while they appear to target the BIR3 domainof XIAP, may have limited bioavailability and therefore limitedtherapeutic application. Moreover, the compounds may not be selectiveagainst other IAPs and indeed other BIR domains, such as BIR2; this lackof specificity may lead to unexpected side effects.

Thus, IAP BIR domains represent an attractive target for the discoveryand development of novel therapeutic agents, especially for thetreatment of proliferative disorders such as cancer.

SUMMARY OF THE INVENTION

We have discovered a novel series of compounds that bind the IAPs andenhance cellular apoptosis through IAP modulation, and which havepharmaceutically acceptable stability and bioavailability. The compoundscause a reduction and/or loss of IAP proteins in cells beforemitochondrial depolarization occurs and prevent the interaction ofcaspase 3, caspase 7, and caspase 9. Hence the results suggest that asmall molecule is capable of down-regulating IAP proteins before celldeath, thus indicating that clinically the use of the compounds mayoffer advantages when administered in combination with other inducers ofapoptosis.

Specifically, we have demonstrated that the compounds bind to the BIR2and BIR3 domain of mammalian XIAP and promote apoptosis of cancer cellsas a single agent or in combination with a chemotherapeutic agent or adeath receptor agonist, such as TRAIL or agonist TRAIL receptorantibodies. Moreover, the compounds were shown to cause reduction ofcellular IAPs from cells which can be blocked by a proteasome inhibitor.Advantageously, the compounds described herein have pro-apoptoticactivity in various cancer cell lines such as bladder, breast,pancreatic, colon, leukemic, lung, lymphoma, multiple myloma andovarian, and may also find application in other cancer cell lines and indiseases where cells are resistant to apoptosis. The compounds werefound to kill cancer cells in a synergistic manner with TRAIL or withagonist TRAIL receptor anti-bodies. These results suggest that compoundsof the instant invention will demonstrate anti-cancer activity againstsolid tumours and tumours originating from the hematologicalmalignancies. Moreover, the compounds of the present invention may alsofind application in preventing cancer cell metastasis, invasion,inflammation, and in other diseases characterized by cells that areresistant to apoptosis. The compounds may also be useful in thetreatment of autoimmune diseases.

According to one aspect embodiment of the present invention, there isprovided an isomer, enantiomer, diastereoisomer or tautomer of acompound represented by Formula I:

or a salt thereof,wherein:

-   n is 0 or 1;-   m is 0, 1 or 2;-   p is 1 or 2;-   Y is NH, O or S;-   A and A¹ are independently selected from    -   1) —CH₂—,    -   2) —CH₂CH₂—,    -   3) —C(CH₃)₂—,    -   4) —CH(C₁-C₆ alkyl),    -   5) —CH(C₃-C₇ cycloalkyl),    -   6) —C₃-C₇ cycloalkyl-,    -   7) —CH(C₁-C₆ alkyl-C₃-C₇ cycloalkyl), or    -   8) —C(O)—;-   B and B¹ are independently C₁-C₆ alkyl;-   BG is    -   1) —X-L-X¹—; or-   BG is-   X and X¹ are independently selected from-   L is selected from:    -   1) —C₁-C₁₀ alkyl-,    -   2) —C₂-C₆ alkenyl-,    -   3) —C₂-C₄ alkynyl-,    -   4) —C₃-C₇ cycloalkyl-,    -   5) -phenyl-,    -   6) -biphenyl-,    -   7) -heteroaryl-,    -   8) -heterocyclyl-,    -   9) —C₁-C₆ alkyl-C₂-C₆ alkenyl)-C₁-C₆ alkyl-,    -   10) —C₁-C₆alkyl-C₂-C₄ alkynyl)-C₁-C₆ alkyl,    -   11) —C₁-C₆ alkyl-C₃-C₇ cycloalkyl)-C₁-C₆ alkyl,    -   12) —C₁-C₆ alkyl-phenyl-C₁-C₆ alkyl,    -   13) —C₁-C₆ alkyl-biphenyl-C₁-C₆ alkyl,    -   14) —C₁-C₆ alkyl-heteroaryl-C₁-C₆ alkyl,    -   15) —C₁-C₆ alkyl-heterocyclyl-C₁-C₆ alkyl, or    -   16) —C₁-C₆ alkyl-O—C₁-C₆ alkyl;-   R¹, R¹⁰⁰, R² and R²⁰⁰ are independently selected from:    -   1) H, or    -   2) C₁-C₆ alkyl optionally substituted with one or more R⁶        substituents;-   Q and Q¹ are each independently    -   1) NR⁴R⁵,    -   2) OR¹¹, or    -   3) S(O)_(m)R¹¹; or-   Q and Q¹ are each independently    wherein G is a 5, 6 or 7 membered ring which optionally incorporates    one or more heteroatoms chosen from S, N or O, the ring being    optionally substituted with one or more R¹² substituents;-   R⁴ and R⁵ are each independently    -   1) H,    -   2) haloalkyl,    -   3) ←C₁-C₆ alkyl,    -   4) ←C₂-C₆ alkenyl,    -   5) ←C₂-C₄ alkynyl,    -   6) ←C₃-C₇ cycloalkyl,    -   7) ←C₃-C₇ cycloalkenyl,    -   8) ←aryl,    -   9) ←heteroaryl,    -   10) ←heterocyclyl,    -   11) ←heterobicyclyl,    -   12) ←C(O)—R¹¹,    -   13) ←C(O)O—R¹¹,    -   14) ←C(═Y)NR⁸R⁹, or    -   15) ←S(O)₂—R¹¹,        wherein the alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl is        optionally substituted with one or more R⁶ substituents; and        wherein the aryl, heteroaryl, heterocyclyl, and heterobicyclyl        is optionally substituted with one or more R¹⁰ substituents;-   R⁶ is    -   1) halogen,    -   2) NO₂,    -   3) CN,    -   4) haloalkyl,    -   5) C₁-C₆ alkyl,    -   6) C₂-C₆ alkenyl,    -   7) C₂-C₄ alkynyl,    -   8) C₃-C₇ cycloalkyl,    -   9) C₃-C₇ cycloalkenyl,    -   10) aryl,    -   11) heteroaryl,    -   12) heterocyclyl,    -   13) heterobicyclyl,    -   14) OR⁷,    -   15) S(O)_(m)R¹,    -   16) NR⁸R⁹,    -   17) NR⁸S(O)₂R¹¹,    -   18) COR⁷,    -   19) C(O)OR⁷,    -   20) CONR⁸R⁹,    -   21) S(O)₂NR⁸R⁹    -   22) OC(O)R⁷,    -   23) OC(O)Y—R¹¹,    -   24) SC(O)R⁷, or    -   25) NC(Y)NR⁸R⁹,        wherein the aryl, heteroaryl, heterocyclyl, and heterobicyclyl        is optionally substituted with one or more R¹⁰ substituents;-   R⁷ is    -   1) H,    -   2) haloalkyl,    -   3) C₁-C₆ alkyl,    -   4) C₂-C₆ alkenyl,    -   5) C₂-C₄ alkynyl,    -   6) C₃-C₇ cycloalkyl,    -   7) C₃-C₇ cycloalkenyl,    -   8) aryl,    -   9) heteroaryl,    -   10) heterocyclyl,    -   11) heterobicyclyl,    -   12) R⁸R⁹NC(═Y), or    -   13) C₁-C₆ alkyl-C₂-C₄ alkenyl, or    -   14) C₁-C₆ alkyl-C₂-C₄ alkynyl,        wherein the alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl is        optionally substituted with one or more R⁶ substituents; and        wherein the aryl, heteroaryl, heterocyclyl, and heterobicyclyl        is optionally substituted with one or more R¹⁰ substituents;-   R⁸ and R⁹ are each independently    -   1) H,    -   2) haloalkyl,    -   3) C₁-C₆ alkyl,    -   4) C₂-C₆ alkenyl,    -   5) C₂-C₄ alkynyl,    -   6) C₃-C₇ cycloalkyl,    -   7) C₃-C₇ cycloalkenyl,    -   8) aryl,    -   9) heteroaryl,    -   10) heterocyclyl,    -   11) heterobicyclyl,    -   12) C(O)R¹¹,    -   13) C(O)Y—R¹¹, or    -   14) S(O)₂—R¹¹,        wherein the alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl is        optionally substituted with one or more R⁶ substituents; and        wherein the aryl, heteroaryl, heterocyclyl, and heterobicyclyl        is optionally substituted with one or more R¹⁰ substituents;-   or R⁸ and R⁹ together with the nitrogen atom to which they are    bonded form a five, six or seven membered heterocyclic ring    optionally substituted with one or more R⁶ substituents;-   R¹⁰ is    -   1) halogen,    -   2) NO₂,    -   3) CN,    -   4) B(OR ¹³)(OR¹⁴),    -   5) C₁-C₆ alkyl,    -   6) C₂-C₆ alkenyl,    -   7) C₂-C₄ alkynyl,    -   8) C₃-C₇ cycloalkyl,    -   9) C₃-C₇ cycloalkenyl,    -   10) haloalkyl,    -   11) OR⁷,    -   12) NR⁸R⁹,    -   13) SR⁷,    -   14) COR⁷,    -   15) C(O)OR⁷,    -   16) S(O)_(m)R⁷,    -   17) CONR⁸R⁹,    -   18) S(O)₂NR⁸R⁹,    -   19) aryl,    -   20) heteroaryl,    -   21 ) heterocyclyl, or    -   22) heterobicyclyl,        wherein the alkyl, alkenyl, alkynyl, cycloalkyl, and        cycloalkenyl is optionally substituted with one or more R⁶        substituents;-   R¹¹ is    -   1) haloalkyl,    -   2) C₁-C₆ alkyl,    -   3) C₂-C₆ alkenyl,    -   4) C₂-C₄ alkynyl,    -   5) C₃-C₇ cycloalkyl,    -   6) C₃-C₇ cycloalkenyl,    -   7) aryl,    -   8) heteroaryl,    -   9) heterocyclyl, or    -   10) heterobicyclyl,        wherein the alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl is        optionally substituted with one or more R⁶ substituents; and        wherein the aryl, heteroaryl, heterocyclyl, and heterobicyclyl        is optionally substituted with one or more R¹⁰ substituents;-   R¹² is    -   1) haloalkyl,    -   2) C₁-C₆ alkyl,    -   3) C₂-C₆ alkenyl,    -   4) C₂-C₄ alkynyl,    -   5) C₃-C₇ cycloalkyl,    -   6) C₃-C₇ cycloalkenyl,    -   7) aryl,    -   8) heteroaryl,    -   9) heterocyclyl,    -   10) heterobicyclyl,    -   11) C(O)—R¹¹,    -   12) C(O)O—R¹¹,    -   13) C(O)NR⁸R⁹,    -   14) S(O)_(m)—R¹¹, or    -   15) C(═Y)NR⁸R⁹,        wherein the alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl is        optionally substituted with one or more R⁶ substituents; and        wherein the aryl, heteroaryl, heterocyclyl, and heterobicyclyl        is optionally substituted with one or more R¹⁰ substituents;-   R¹³ and R¹⁴ are each independently    -   1) H, or    -   2) C₁-C₆ alkyl; or-   R¹³ and R¹⁴ are combined to form a heterocyclic ring or a    heterobicyclyl ring;    or a prodrug; or the compound of Formula I is labeled with a    detectable label or an affinity tag.

According to one alternative aspect of the present invention, there isprovided a compound, according to Formula 2:

wherein n, R¹, R², R¹⁰⁰, R²⁰⁰, A, A¹, Q, Q¹, B, B¹, and BG as definedabove;wherein the dotted line represents a hypothetical dividing line forcomparing the substituents associated with M1 and M2.

In another aspect of the present invention, M1 is the same as M2.

In another aspect of the present invention, M1 is different from M2.

In one aspect of the present invention, there is provided anintermediate compound represented by Formula 2(iii):

wherein PG² is a protecting group, and R¹, R², B, A, and Q are asdefined herein.

In another aspect of the present invention, there is provided anintermediate compound represented by Formula 3(iii):

wherein B, B¹, A, A¹, Q and Q¹ are as defined herein.

In another aspect of the present invention, there is provided anintermediate compound represented by Formula 4(iii):

wherein PG³ is a protecting group, and B, R¹, R², A, and Q are asdefined herein.

In another aspect of the present invention, there is provided anintermediate compound represented by Formula 5(i):

wherein PG³ are protecting groups, and B, B¹, R¹, R¹⁰⁰, R², R²⁰⁰ , A,A¹, Q and Q¹ are as defined herein.

In another aspect of the present invention, there is provided anintermediate compound represented by Formula 6(iii):

wherein PG³ is a protecting group, and R¹, R², B, A, and Q are asdefined herein.

In another aspect of the present invention, there is provided anintermediate compound represented by Formula 7(iii):

wherein PG³ is a protecting group, and R¹, R², B, A, and Q are asdefined herein.

In another aspect of the present invention, there is provided anintermediate compound represented by Formula 8(iii):

wherein B, B¹, A, A¹, Q and Q¹ are as defined herein.

In another aspect of the present invention, there is provided a processfor producing compounds represented by Formula I, described hereinabove,the process comprising:

-   -   a) coupling two intermediates represented by Formula 2(iii):        in a solvent; and    -   b) removing the protecting groups so as to form compounds of        Formula 1.

In another aspect of the present invention, there is provided a processfor producing compounds represented by Formula I, described hereinabove,the process comprising:

-   -   a) coupling an intermediate represented by Formula 3(iii):        in a solvent; and    -   b) removing the protecting groups so as to form compounds of        Formula 1.

In another aspect of the present invention, there is provided a processfor producing compounds represented by Formula I, described herein, theprocess comprising:

-   -   a) coupling an intermediate represented by Formula 4(iii):        and an activated diacid, such as a diacid chloride or a diacid        activated using 2 equiv of peptide coupling agents, in a        solvent; and    -   b) removing the protecting groups so as to form compounds of        Formula 1.

In another aspect of the present invention, there is provided a processfor producing compounds represented by Formula I, described herein, theprocess comprising:

-   -   a) coupling two intermediates represented by Formula 4(iii):        with triphosgene, or a triphosgene equivalent, in a solvent; and    -   b) removing the protecting groups so as to form compounds of        Formula 1.

In another aspect of the present invention, there is provided a processfor producing compounds represented by Formula I, described herein, theprocess comprising:

-   -   a) coupling two intermediates represented by Formula 4(iii):        with oxalyl chloride in a solvent; and    -   b) removing the protecting groups so as to form compounds of        Formula 1.

In another aspect of the present invention, there is provided a processfor producing compounds represented by Formula I, described herein, theprocess comprising:

-   -   a) coupling an intermediate represented by Formula 6(iii):        and either a bis-acid chloride or a bis-acid, using a coupling        agent, in a solvent; and    -   b) removing the protecting groups so as to form compounds of        Formula 1.

In another aspect of the present invention, there is provided a processfor producing compounds represented by Formula I, described herein, theprocess comprising:

-   -   a) coupling an intermediate represented by Formula 7(iii):        and a diamine using a coupling agent in a solvent; and    -   b) removing the protecting groups so as to form compounds of        Formula 1.

In another aspect of the present invention, there is provided a processfor producing compounds represented by Formula I, described hereinabove,the process comprising:

-   -   a) coupling an intermediate represented by Formula 8(iii):        in a solvent; and    -   b) removing the protecting groups so as to form compounds of        Formula 1.

In another aspect of the present invention, there is provided a processfor producing compounds represented by Formula I, described hereinabove,the process comprising:

-   -   a) hydrogenation of a compound represented by 1g        in a solvent,    -   b) filtration and concentration of the solvent to provide a        compound of formula 1q.

In another aspect of the present invention, there is provided apharmaceutical composition comprising a compound, as described above,mixed with a pharmaceutically acceptable carrier, diluent or excipient.

In another aspect of the present invention, there is provided apharmaceutical composition adapted for administration as an agent fortreating a proliferative disorder in a subject, comprising atherapeutically effective amount of a compound, as described above.

In another aspect of the present invention, there is provided apharmaceutical composition comprising a compound of Formula I incombination with one or more death receptor agonists, for example, anagonist of TRAIL receptor.

In another aspect of the present invention, there is provided apharmaceutical composition comprising a compound of formula I incombination with any therapeutic agent that increases the response ofone or more death receptor agonists, for example cytotoxic cytokinessuch as interferons.

In another aspect of the present invention, there is provided a methodof preparing a pharmaceutical composition, the method comprising: mixinga compound, as described above, with a pharmaceutically acceptablecarrier, diluent or excipient.

In another aspect of the present invention, there is provided a methodof treating a disease state characterized by insufficient apoptosis, themethod comprising: administering to a subject in need thereof, atherapeutically effective amount of a pharmaceutical composition, asdescribed above, so as to treat the disease state.

In another aspect of the present invention, there is provided a methodof modulating IAP function, the method comprising: contacting a cellwith a compound of the present invention so as to prevent binding of aBIR binding protein to an IAP BIR domain thereby modulating the IAPfunction.

In another aspect of the present invention, there is provided a methodof treating a proliferative disease, the method comprising:administering to a subject in need thereof, a therapeutically effectiveamount of the pharmaceutical composition, as described above, so as totreat the proliferative disease.

In another aspect of the present invention, there is provided a methodof treating cancer, the method comprising: administering to a subject inneed thereof, a therapeutically effective amount of the pharmaceuticalcomposition, as described above, so as to treat the cancer.

In another aspect of the present invention, there is provided a methodof treating cancer, the method comprising: administering to the subjectin need thereof, a therapeutically effective amount of a pharmaceuticalcomposition, as described above, in combination or sequentially with anagent selected from:

-   a) an estrogen receptor modulator,-   b) an androgen receptor modulator,-   c) retinoid receptor modulator,-   d) a cytotoxic agent,-   e) an antiproliferative agent,-   f) a prenyl-protein transferase inhibitor,-   g) an HMG-CoA reductase inhibitor,-   h) an HIV protease inhibitor,-   i) a reverse transcriptase inhibitor,-   k) an angiogenesis inhibitor,-   l) a PPAR-.γ agonist,-   m) a PPAR-.δ. agonist,-   n) an inhibitor of inherent multidrug resistance,-   o) an anti-emetic agent,-   p) an agent useful in the treatment of anemia,-   q) agents useful in the treatment of neutropenia,-   r) an immunologic-enhancing drug.-   s) a proteasome inhibitor;-   t) an HDAC inhibitor;-   u) an inhibitor of the chemotrypsin-like activity in the proteasome;    or-   v) E3 ligase inhibitors;-   w) a modulator of the immune system such as, but not limited to,    interferon-alpha, Bacillus Calmette-Guerin (BCG), and ionizing    radiation (UVB) that can induce the release of cytokines, such as    the interleukins, TNF, or induce release of death receptor ligands    such as TRAIL;-   x) a modulator of death receptors TRAIL and TRAIL agonists such as    the humanized antibodies HGS-ETR1 and HGS-ETR2;    or in combination or sequentially with radiation therapy, so as to    treat the cancer.

In another aspect of the present invention, there is provided a methodfor the treatment or prevention of a proliferative disorder in asubject, the method comprising: administering to the subject atherapeutically effective amount of the composition, described above.

In another aspect of the present invention, the method further comprisesadministering to the subject a therapeutically effective amount of achemotherapeutic agent prior to, simultaneously with or afteradministration of the composition.

In yet another aspect, the method further comprises administering to thesubject a therapeutically effective amount of a death receptor agonistprior to, simultaneously with or after administration of thecomposition. The death receptor agonist is TRAIL or the death receptoragonist is a TRAIL antibody. The death receptor agonist is typicallyadministered in an amount that produces a synergistic effect.

In another aspect of the present invention, there is provided a probe,the probe being a compound of Formula I above, the compound beinglabeled with a detectable label or an affinity tag.

In another aspect of the present invention, there is provided a methodof identifying compounds that bind to an IAP BIR domain, the assaycomprising:

-   -   a) contacting an IAP BIR domain with a probe to form a probe:BIR        domain complex, the probe being displaceable by a test compound;    -   b) measuring a signal from the probe so as to establish a        reference level;    -   c) incubating the probe:BIR domain complex with the test        compound;    -   d) measuring the signal from the probe;    -   e) comparing the signal from step d) with the reference level, a        modulation of the signal being an indication that the test        compound binds to the BIR domain,        wherein the probe is a compound of Formula I labeled with a        detectable label or an affinity label.

BRIEF DESCRIPTION OF THE DRAWINGS

Further aspects and advantages of the present invention will becomebetter understood with reference to the description in association withthe following Figure, wherein:

FIG. 1 is a graph illustrating a combination anti-cancer therapy in vivoin which compound 23 showed an increasing anti-tumor effect incombination with mitomycin-C with increasing dose, with 5 mg/kg showingsuperior anti-tumor effects compared to the 1 mg/kg dose.

DETAILED DESCRIPTION OF THE INVENTION

In many cancer and other diseases, an up-regulation of IAP induced bygene defects or by chemotherapeutic agents has been correlated to anincreased resistance to apoptosis. Interestingly our results show thatcells decreased in IAPs level are more sensitive to TRAIL inducedapoptosis. It is believed that a small molecule, which will induce IAPloss from disease cells, will be useful as a therapeutic agent. Wereport herein compounds that can directly bind to IAPs, cause a downregulation of the IAP proteins in cell before cell death, induceapoptosis in cancer cells, and have a synergistic effect in combinationwith inducers of apoptosis. This may provide clinical advantages interms of the selectivity of therapy based on the phenotype of the cancercells. Also advantageous would be the use of the compounds of thepresent invention in combination therapy with other agents in terms ofthe doses of administration and the time of scheduling the doses.

The compounds of the present invention are useful as BIR domain bindingcompounds in mammalian IAPs and are represented by Formula I. Thefollowing are embodiments, groups and substituents of the compoundsaccording to Formula I, which are described hereinafter in detail.

n:

In one subset of compounds of Formula 1, n is 1.

Any and each individual definition of n as set out herein may becombined with any and each individual definition of Core, R¹, R², R¹⁰⁰,R²⁰⁰, A, A¹, Q, Q¹, B, B¹, and BG as set out herein.

A and A¹:

In one subset of compounds of Formula 1, A and A¹ are both CH₂.

In an alternative subset of compounds of Formula 1, A and A¹ are bothC═O.

In another alternative subset of compounds of Formula 1, A is CH₂ and A¹is C═O.

Any and each individual definition of A and A¹ as set out herein may becombined with any and each individual definition of Core, n, R¹, R²,R¹⁰⁰, R²⁰⁰, Q, Q¹, B, B¹, and BG as set out herein.

Core:

Therefore, the present invention comprises compounds of Formula 1athrough 1c:

wherein BG, B, B¹, Q, Q¹, R¹, R¹⁰⁰, R² and R²⁰⁰ are as definedhereinabove and hereinafter.

In one example, the present invention comprises compounds of Formula 1a.

In an alternative example, the present invention comprises compounds ofFormula 1b.

Any and each individual definition of Core as set out herein may becombined with any and each individual definition of A, A¹, n, R¹, R²,R¹⁰⁰, R²⁰⁰, Q, Q¹, B, B¹, and BG as set out herein.

B and B¹:

In one subset of the aforesaid compounds, B and B¹ are both C₁-C₄ alkyl.

Any and each individual definition of B and B¹ as set out herein may becombined with any and each individual definition of Core, A, A¹, n, R¹,R², R¹⁰⁰, R², Q, Q¹, and BG as set out herein.

BG:

In one subset of the aforesaid compounds, BG is —X-L-X¹—.

Therefore the invention comprises compounds of Formula 1d and 1e:

wherein L, B, B¹, X, X¹, Q, Q¹, R¹, R¹⁰⁰, R² and R²⁰⁰ are as definedherein.

In an alternative subset of the aforesaid compounds, BG is

Therefore, the invention alternatively comprises compounds of Formula 1for 1g:

wherein A, A¹, B, B¹, Q, Q¹, R¹, R¹⁰⁰, R² and R²⁰⁰ are as definedherein.

Any and each individual definition of BG as set out herein may becombined with any and each individual definition of Core, A, A¹, n, R¹,R², R¹⁰⁰, R²⁰⁰, Q, Q¹, B, and B¹ as set out herein.

X and X¹:

In one subset of the aforesaid compounds, X and X¹ are independentlyselected from

In another subset of the aforesaid compounds, X and X¹ are independentlyselected from:

Typical examples of X and X¹ include both X and X¹ as being O,

Any and each individual definition of X and X¹ as set out herein may becombined with any and each individual definition of Core, A, A¹, n, R¹,R², R¹⁰⁰, R²⁰⁰, Q, Q¹, B, B¹, and BG as set out herein.

L:

In one subset of the aforesaid compounds, L is selected from:

-   -   1) —C₁-C₁₀ alkyl-,    -   2) —C₂-C₄ alkynyl-,    -   3) -phenyl-,    -   4) -biphenyl-,    -   5) —C₁-C₆ alkyl-(C₂-C₄ alkynyl)-C₁-C₆ alkyl,    -   6) —C₁-C₆ alkyl-phenyl-C₁-C₆ alkyl,    -   7) —C₁-C₆ alkyl-biphenyl-C₁-C₆ alkyl, or    -   8) —C₁-C₆ alkyl-O—C₁-C₆ alkyl.

In another subset of the aforesaid compounds, L is selected from

-   -   1) —C₁-C₁₀ alkyl-,    -   2) -phenyl-,    -   3) -biphenyl-,    -   4) —CH₂—(C₂-C₄ alkynyl)-CH₂—,    -   5) —CH₂-phenyl-CH₂—,    -   6) —CH₂-biphenyl-CH₂—, or    -   7) —C₁-C₆ alkyl-O—C₁-C₆ alkyl.

Typical examples of L include

Any and each individual definition of L as set out herein may becombined with any and each individual definition of Core, A, A¹, n, R¹,R², R¹⁰⁰, R²⁰⁰, Q, Q¹, B, and B¹ as set out herein.

r:

In the aforesaid aspect, r is an integer of 1, 2, 3, 4, 5, 6, 7, or 8.

Any and each individual definition of r as set out herein may becombined with any and each individual definition of Core, A, A¹, n, R¹,R², R¹⁰⁰, R²⁰⁰, Q, Q¹, B, and B¹ as set out herein.

More explicitly, the invention comprises compounds of Formulae 1h, 1i,1j, 1k, 1l, and 1m:

wherein B, B¹, X, X¹, Q, Q¹, R¹, R¹⁰⁰, R² and R²⁰⁰ are as definedherein.

R¹ and R¹⁰⁰:

In one subset of the aforesaid compounds R¹ and R¹⁰⁰ are both C₁-C₆alkyl.

In one example, R¹ and R¹⁰⁰ are both CH₃.

Any and each individual definition of R¹ and R¹⁰⁰ as set out herein maybe combined with any and each individual definition of Core, A, A¹, n,R², R²⁰⁰, Q, Q¹, B, B¹, and BG as set out herein.

R² and R²⁰⁰:

In one subset of the aforesaid compounds R² and R²⁰⁰ are both C₁-C₆alkyl.

In one example, R² and R²⁰⁰ are both CH₃.

Any and each individual definition of R² and R²⁰⁰ as set out herein maybe combined with any and each individual definition of Core, A, A¹, n,R¹, R¹⁰⁰, Q, Q¹, B, B¹, and BG as set out herein.

Q and Q¹:

In one subset of the aforesaid compounds, Q and Q¹ are both NR⁴R⁵,wherein R⁴ and R⁵ are as defined herein.

Any and each individual definition of Q and Q¹ as set out herein may becombined with any and each individual definition of Core, A, A¹, n, R¹,R¹⁰⁰, R², R²⁰⁰, B, B¹, and BG as set out herein.

R⁴ and R⁵:

In one subset of the aforesaid compounds in which A and A¹ are both C═O,R⁴ is H and R⁵ is selected from

-   -   1) haloalkyl,    -   2) ←C₁-C₆ alkyl,    -   3) ←C₂-C₆ alkenyl,    -   4) ←C₂-C₄ alkynyl,    -   5) ←C₃-C₇ cycloalkyl,    -   6) ←C₃-C₇ cycloalkenyl,    -   7) ←aryl,    -   8) ←heteroaryl,    -   9) ←heterocyclyl, or    -   10) ←heterobicyclyl,        wherein the alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl is        optionally substituted with one or more R⁶ substituents; and        wherein the aryl, heteroaryl, heterocyclyl, and heterobicyclyl        is optionally substituted with one or more R¹⁰ substituents;        wherein R⁶ and R¹⁰ are as defined herein.

In another subset of the above compounds, R⁴ is H and R⁵ is selectedfrom:

-   -   1) ←C₃-C₇ cycloalkyl,    -   2) ←C₃-C₇ cycloalkenyl,    -   3) ←aryl,    -   4) ←heteroaryl,    -   5) ←heterocyclyl, or    -   6) ←heterobicyclyl.

In still another subset of the above compounds, R⁴ is H and R⁵ is aryl.

In one example, R⁴ is H and R⁵ is

Therefore, when A and A¹ are both C═O, then Q and Q¹ are both

In an alternative subset of the aforesaid compounds in which A and A¹are both CH₂, then R⁴ and R⁵ are each independently

-   -   1) H,    -   2) haloalkyl,    -   3) ←C₁-C₆ alkyl,    -   4) ←C₂-C₆ alkenyl,    -   5) ←C₂-C₄ alkynyl,    -   6) ←C₃-C₇ cycloalkyl,    -   7) ←C₃-C₇ cycloalkenyl,    -   8) ←aryl,    -   9) ←heteroaryl,    -   10) ←heterocyclyl,    -   11) ←heterobicyclyl,    -   12) ←C(O)—R¹¹,    -   13) ←C(O)O—R¹¹,    -   13) ←C(═Y)NR⁸R⁹, or    -   14) ←S(O)₂—R¹¹,        wherein the alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl is        optionally substituted with one or more R⁶ substituents; and        wherein the aryl, heteroaryl, heterocyclyl, and heterobicyclyl        is optionally substituted with one or more R¹⁰ substituents;        wherein Y, R⁶, R⁸, R⁹, R¹⁰ and R¹¹ are as defined herein.

In another subset of the above compounds, R⁴ and R⁵ are independentlyselected from

-   -   1) H,    -   2) C₁-C₆ alkyl,    -   3) ←C(O)—R¹¹,    -   4) ←C(O)O—R¹¹, or    -   5) ←S(O)₂—R¹¹,        wherein the alkyl is substituted with an R⁶ substituent;        wherein R⁶, and R¹¹ are as defined herein.

In one subset of the aforesaid compounds,

-   R⁴ is    -   1) H,    -   2) ←C(O)—R¹¹,    -   3) ←C(O)O—R¹¹, or    -   4) ←S(O)₂—R¹¹; and-   R⁵ is C₁-C₆ alkyl substituted with a phenyl;    wherein R¹¹ is as defined herein.

In another subset of the aforesaid compounds,

-   R⁴ is    -   1) H,    -   2) ←C(O)—R¹¹,    -   3) ←C(O)O—R¹¹, or    -   4) ←S(O)₂—R¹¹; and-   R⁵ is    wherein R¹¹ is as defined herein.

Any and each individual definition of R⁴ and R⁵ as set out herein may becombined with any and each individual definition of Core, A, A¹, n, R¹,R¹⁰⁰, R², R²⁰⁰, B, B¹, and BG as set out herein.

R¹¹:

In one subset of the aforesaid compounds,

-   R¹¹ is    -   1) haloalkyl,    -   2) C₁-C₆ alkyl,    -   3) C₂-C₆ alkenyl,    -   4) C₂-C₄ alkynyl,    -   5) aryl,    -   6) heteroaryl,    -   7) heterocyclyl, or    -   8) heterobicyclyl,        wherein the alkyl, alkenyl, alkynyl is optionally substituted        with one or more R⁶ substituents; and wherein the aryl,        heteroaryl, heterocyclyl, and heterobicyclyl is optionally        substituted with one or more R¹⁰ substituents;        wherein R⁶ and R¹⁰ are as defined herein.

In another subset of the aforesaid compounds, R¹¹ is

-   -   1) haloalkyl,    -   2) C₁-C₆ alkyl,    -   3) aryl,    -   4) heteroaryl, or    -   5) heterocyclyl,        wherein the alkyl is optionally substituted with one or two R⁶        substituents; and wherein the aryl, heteroaryl and heterocyclyl        is substituted with one R¹⁰ substituent;        wherein R⁶ and R¹⁰ are as defined herein.

In one subset of the aforesaid compounds, R¹¹ is

-   -   1) haloalkyl,    -   2) C₁-C₆ alkyl optionally substituted with one or two R⁶        substituents, or    -   3) phenyl optionally substituted with one R¹⁰ substituent;        wherein the R⁶ and the R¹⁰ substituents are as defined herein.

Any and each individual definition of R¹¹ as set out herein may becombined with any and each individual definition of Core, A, A¹, n, R¹,R¹⁰⁰, R², R²⁰⁰, R⁴, R⁵, B, B¹, and BG as set out herein.

R⁶:

In one subset of the aforesaid compounds, R⁶ is

-   -   1) halogen,    -   2) NO₂,    -   3) CN,    -   4) aryl,    -   5) heteroaryl,    -   6) heterocyclyl,    -   7) heterobicyclyl,    -   8) OR⁷,    -   9) SR⁷, or    -   10) NR⁸R⁹,        wherein the aryl, heteroaryl, heterocyclyl, and heterobicyclyl        is optionally substituted with one or more R¹⁰ substituents;        wherein R⁷, R⁸, R⁹ and R¹⁰ are as defined herein.

In another subset of the aforesaid compounds, R⁶ is

-   -   1) halogen,    -   2) aryl, or    -   3) NR⁸R⁹,        wherein the aryl is optionally substituted with one R¹⁰        substituent;        wherein R⁸, R⁹ and R¹⁰ are as defined herein.

In one subset of the aforesaid compounds, R⁶ is

-   -   1) halogen,    -   2) phenyl, or    -   3) NR⁸R⁹,        wherein the phenyl is optionally substituted with one R¹⁰        substituent;        wherein R⁸ and R⁹ are as defined herein.

Any and each individual definition of R⁶ as set out herein may becombined with any and each individual definition of Core, A, A¹, n, R¹,R¹⁰⁰, R², R²⁰⁰, R⁴, R⁵, B, B¹, and BG as set out herein.

R⁸ and R⁹:

In one subset of the aforesaid compounds, R⁸ and R⁹ are eachindependently

-   -   1) H,    -   2) haloalkyl,    -   3) C₁-C₆ alkyl,    -   4) C₂-C₆ alkenyl,    -   5) C₂-C₄ alkynyl,    -   6) C₃-C₇ cycloalkyl, or    -   7) C₃-C₇ cycloalkenyl,        wherein the alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl is        optionally substituted with one or more R⁶ substituents;        wherein the R⁶ substituents are as defined herein.

In another subset of the aforesaid compounds, R⁸ and R⁹ are eachindependently

-   -   1) H, or    -   2) C₁-C₆ alkyl,        wherein the alkyl is optionally substituted with an aryl.

Any and each individual definition of R⁸ and R⁹ as set out herein may becombined with any and each individual definition of Core, A, A¹, n, R¹,R¹⁰⁰, R², R²⁰⁰, R⁴, R⁵, B, B¹, and BG as set out herein.

R¹⁰:

In one aspect of the aforesaid compounds, R¹⁰ is

-   -   1) halogen,    -   2) NO₂,    -   3) CN,    -   4) haloalkyl,    -   5) OR⁷,    -   6) NR⁸R⁹, or    -   7) SR⁷;        wherein R⁷, R⁸, and R⁹ are as defined herein.

In another aspect of the aforesaid compounds, R¹⁰ is

-   -   1) halogen, or    -   2) OC₁-C₆ alkyl.

Any and each individual definition of R¹⁰ as set out herein may becombined with any and each individual definition of Core, A, A¹, n, R¹,R¹⁰⁰, R², R²⁰⁰, R⁴, R⁵, B, B¹, and BG as set out herein.

Thus, when A and A¹ are both CH₂, then Q and Q¹ are independentlyselected from:

The present invention also encompasses an isomer, enantiomer,diastereoisomer or tautomer of a compound represented by Formula I:

or a salt thereof,wherein:

-   n is 1;-   m is 0, 1 or 2;-   Y is NH, O or S;-   A and A¹ are independently selected from    -   1) —CH₂—, or    -   2) —C(O)—;-   B and B¹ are independently C₁-C₆ alkyl;-   BG is    -   1) —X-L-X¹—; or-   BG is-   X and X¹ are independently selected from-   L is selected from:    -   1) —C₁-C₁₀ alkyl-,    -   2) —C₂-C₆ alkenyl-,    -   3) —C₂-C₄ alkynyl-,    -   4) —C₃-C₇ cycloalkyl-,    -   5) -phenyl-,    -   6) -biphenyl-,    -   7) -heteroaryl-,    -   8) -heterocyclyl-,    -   9) —C₁-C₆ alkyl-(C₂-C₆ alkenyl)-C₁-C₆ alkyl-,    -   10) —C₁-C₆ alkyl-C₂-C₄ alkynyl)-C₁-C₆ alkyl,    -   11) —C₁-C₆ alkyl-(C₃-C₇ cycloalkyl)-C₁-C₆ alkyl,    -   12) —C₁-C₆ alkyl-phenyl-C₁-C₆ alkyl,    -   13) —C₁-C₆ alkyl-biphenyl-C₁-C₆ alkyl,    -   14) —C₁-C₆ alkyl-heteroaryl-C₁-C₆ alkyl,    -   15) —C₁-C₆ alkyl heterocyclyl-C₁-C₆ alkyl, or    -   16) —C₁-C₆ alkyl-O—C₁-C₆ alkyl;-   R¹, R¹⁰⁰, R² and R²⁰⁰ are independently selected from:    -   1) H, or    -   2) C₁-C₆ alkyl optionally substituted with one or more R⁶        substituents;-   Q and Q¹ are each independently NR⁴R⁵;-   R⁴ and R⁵ are each independently    -   1) H,    -   2) haloalkyl,    -   3) ←C₁-C₆ alkyl,    -   4) ←C₂-C₆ alkenyl,    -   5) ←C₂-C₄ alkynyl,    -   6) ←C₃-C₇ cycloalkyl,    -   7) ←C₃-C₇ cycloalkenyl,    -   8) ←aryl,    -   9) ←heteroaryl,    -   10) ←heterocyclyl,    -   11) ←heterobicyclyl,    -   12) ←C(O)—R¹¹,    -   13) ←C(O)O—R¹¹,    -   14) ←C(═Y)NR⁸R⁹, or    -   15) ←S(O)₂—R¹¹,        wherein the alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl is        optionally substituted with one or more R⁶ substituents; and        wherein the aryl, heteroaryl, heterocyclyl, and heterobicyclyl        is optionally substituted with one or more R¹⁰ substituents;-   R⁶is    -   1) halogen,    -   2) NO₂,    -   3) CN,    -   4) haloalkyl,    -   5) C₁-C₆ alkyl,    -   6) C₂-C₆ alkenyl,    -   7) C₂-C₄ alkynyl,    -   8) C₃-C₇ cycloalkyl,    -   9) C₃-C₇ cycloalkenyl,    -   10) aryl,    -   11) heteroaryl,    -   12) heterocyclyl,    -   13) heterobicyclyl,    -   14) OR⁷,    -   15) S(O)_(m)R⁷,    -   16) NR⁸R⁹,    -   17) NR⁸S(O)₂R¹¹,    -   18) COR⁷,    -   19) C(O)OR⁷,    -   20) CONR⁸R⁹,    -   21) S(O)₂NR⁸R⁹,    -   22) OC(O)R⁷,    -   23) OC(O)Y—R¹¹,    -   24) SC(O)R⁷, or    -   25) NC(Y)NR⁸R⁹,        wherein the aryl, heteroaryl, heterocyclyl, and heterobicyclyl        is optionally substituted with one or more R¹⁰ substituents;-   R⁷ is    -   1) H,    -   2) haloalkyl,    -   3) C₁-C₆ alkyl,    -   4) C₂-C₆ alkenyl,    -   5) C₂-C₄ alkynyl,    -   6) C₃-C₇ cycloalkyl,    -   7) C₃-C₇ cycloalkenyl,    -   8) aryl,    -   9) heteroaryl,    -   10) heterocyclyl,    -   11) heterobicyclyl,    -   12) R⁸R⁹NC(═Y), or    -   13) C₁-C₆ alkyl-C₂-C₄ alkenyl, or    -   14) C₁-C₆ alkyl-C₂-C₄ alkynyl,        wherein the alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl is        optionally substituted with one or more R⁶ substituents; and        wherein the aryl, heteroaryl, heterocyclyl, and heterobicyclyl        is optionally substituted with one or more R¹⁰ substituents;-   R⁸ and R⁹ are each independently    -   1) H,    -   2) haloalkyl,    -   3) C₁-C₆ alkyl,    -   4) C₂-C₆ alkenyl,    -   5) C₂-C₄ alkynyl,    -   6) C₃-C₇ cycloalkyl,    -   7) C₃-C₇ cycloalkenyl,    -   8) aryl,    -   9) heteroaryl,    -   10) heterocyclyl,    -   11) heterobicyclyl,    -   12) C(O)R¹¹,    -   13) C(O)Y—R¹¹, or    -   14) S(O)₂—R¹¹,        wherein the alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl is        optionally substituted with one or more R⁶ substituents; and        wherein the aryl, heteroaryl, heterocyclyl, and heterobicyclyl        is optionally substituted with one or more R¹⁰ substituents;-   or R⁸ and R⁹ together with the nitrogen atom to which they are    bonded form a five, six or seven membered heterocyclic ring    optionally substituted with one or more R⁶ substituents;-   R¹⁰ is    -   1) halogen,    -   2) NO₂,    -   3) CN,    -   4) B(OR¹³)(OR¹⁴),    -   5) C₁-C₆ alkyl,    -   6) C₂-C₆ alkenyl,    -   7) C₂-C₄ alkynyl,    -   8) C₃-C₇ cycloalkyl,    -   9) C₃-C₇ cycloalkenyl,    -   10) haloalkyl,    -   11) OR⁷,    -   12) NR⁸R⁹,    -   13) SR⁷,    -   14) COR⁷,    -   15) C(O)OR⁷,    -   16) S(O)_(m)R⁷,    -   17) CONR⁸R⁹,    -   18) S(O)₂NR⁸R⁹,    -   19) aryl,    -   20) heteroaryl,    -   21) heterocyclyl, or    -   22) heterobicyclyl,        wherein the alkyl, alkenyl, alkynyl, cycloalkyl, and        cycloalkenyl is optionally substituted with one or more R⁶        substituents; and-   R¹¹ is    -   1) haloalkyl,    -   2) C₁-C₆ alkyl,    -   3) C₂-C₆ alkenyl,    -   4) C₂-C₄ alkynyl,    -   5) C₃-C₇ cycloalkyl,    -   6) C₃-C₇ cycloalkenyl,    -   7) aryl,    -   8) heteroaryl,    -   9) heterocyclyl, or    -   10) heterobicyclyl,        wherein the alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl is        optionally substituted with one or more R⁶ substituents; and        wherein the aryl, heteroaryl, heterocyclyl, and heterobicyclyl        is optionally substituted with one or more R¹⁰ substituents;        or a prodrug; or the compound of Formula I is labeled with a        detectable label or an affinity tag.

In one subset of the compounds of Formula 1, specifically compounds ofFormula 1b, wherein

-   n=1;-   A and A¹ are both C═O,-   B and B¹ are independently C₁-C₄ alkyl;-   BG is —X-L-X¹; or-   BG is-   X and X¹ are independently selected from-   L is selected from    -   1) —C₁-C₁₀ alkyl-,    -   2) -phenyl-,    -   3) -biphenyl-,    -   4) —CH₂—(C₂-C₄ alkynyl)-CH₂—,    -   5) —CH₂-phenyl-CH₂—,    -   6) —CH₂-biphenyl-CH₂—, or    -   7) —C₁-C₆ alkyl-O—C₁-C₆ alkyl;-   R¹, R¹⁰⁰, R² and R²⁰⁰ are each independently CH₃;-   Q and Q¹ are both NR⁴R⁵;-   R⁴ is H; and-   R⁵ is selected from:    -   1) ←C₃-C₇ cycloalkyl,    -   2) ←C₃-C₇ cycloalkenyl,    -   3) ←aryl,    -   4) ←heteroaryl,    -   5) ←heterocyclyl, or    -   6) ←heterobicyclyl.

In another subset of the compounds described above,

-   A and A¹ are both C═O,-   B and B¹ are independently C₁-C₄ alkyl;-   BG is —X-L-X¹; or-   BG is-   X and X¹ are both-   L is-   R¹, R¹⁰⁰, R² and R²⁰⁰ are each independently CH₃;-   Q and Q¹ are both NR⁴R⁵;-   R⁴ is H; and-   R⁵ is

In an alternative subset of the compounds of Formula 1, specificallycompounds of Formula 1a, wherein

-   n=1;-   A and A¹ are both CH₂;-   B and B¹ are independently C₁-C₄ alkyl;-   BG is —X-L-X¹; or-   BG is-   X and X¹ are independently selected from-   L is selected from    -   1) —C₁-C₁₀ alkyl-,    -   2) -phenyl-,    -   3) -biphenyl-,    -   4) —CH₂—(C₂-C₄ alkynyl)-CH₂—,    -   5) —CH₂-phenyl-CH₂—,    -   6) —CH₂-biphenyl-CH₂—, or    -   7) —C₁-C₆ alkyl-O—C₁-C₆ alkyl;-   R¹, R¹⁰⁰, R² and R²⁰⁰ are each independently CH₃;-   Q and Q¹ are both NR⁴R⁵;-   R⁴ is    -   1) H,    -   2) ←C(O)—R¹¹,    -   3) ←C(O)O—R¹¹, or    -   4) ←S(O)₂—R¹¹; and-   R⁵ is C₁-C₆ alkyl substituted with a phenyl;    wherein R¹¹ is as defined herein;-   R¹¹ is    -   1) haloalkyl,    -   2) C₁-C₆ alkyl,    -   3) aryl,    -   4) heteroaryl, or    -   5) heterocyclyl,        wherein the alkyl is optionally substituted with one or two R⁶        substituents; and wherein the aryl, heteroaryl and heterocyclyl        is substituted with one R¹⁰ substituent;        wherein R⁶ and R¹⁰ are as defined herein;-   R⁶ is    -   1) halogen,    -   2) aryl, or    -   3) NR⁸R⁹,        wherein the aryl is optionally substituted with one R¹⁰        substituent;        wherein R⁸, R⁹ and R¹⁰ are as defined herein;-   R⁸ and R⁹ are each independently    -   1) H,    -   2) haloalkyl,    -   3) C₁-C₆ alkyl,    -   4) C₂-C₆ alkenyl,    -   5) C₂-C₄ alkynyl,    -   6) C₃-C₇ cycloalkyl, or    -   7) C₃-C₇ cycloalkenyl,        wherein the alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl is        optionally substituted with one or more R⁶ substituents;        wherein the R⁶ substituents are as defined herein; and-   R¹⁰ is    -   1) halogen,    -   2) NO₂,    -   3) CN,    -   4) haloalkyl,    -   5) OR⁷,    -   6) NR⁸R⁹, or    -   7) SR⁷;        wherein R⁷, R⁸, and R⁹ are as defined herein.

In another subset of the aforesaid compounds,

-   n=1;-   A and A¹ are both CH₂;-   B and B¹ are independently C₁-C₄ alkyl;-   BG is —X-L-X¹; or-   BG is-   X and X¹ are independently selected from-   L is selected from    -   1) —C₁-C₁₀ alkyl-,    -   2) -phenyl-,    -   3) -biphenyl-,    -   4) —CH₂—(C₂-C₄ alkynyl)-CH₂—,    -   5) —CH₂-phenyl-CH₂—,    -   6) —CH₂-biphenyl-CH₂—, or    -   7) —C₁-C₆ alkyl-O—C₁-C₆ alkyl;-   R¹, R¹⁰⁰, R² and R²⁰⁰ are each independently CH₃;-   Q and Q¹ are both NR⁴R⁵;-   R⁴ is    -   1) H,    -   2) ←C(O)—R¹¹,    -   3) ←C(O)O—R¹¹, or    -   4) ←S(O)₂—R¹¹; and-   R⁵ is    wherein R¹¹ is as defined herein;-   R¹¹ is    -   1) haloalkyl,    -   2) C₁-C₆ alkyl optionally substituted with one or two R⁶        substituents, or    -   3) phenyl optionally substituted with one R¹⁰ substituent;        wherein the R⁶ and the R¹⁰ substituents are as defined herein;-   R⁶is    -   1) halogen,    -   2) phenyl, or    -   3) NR⁸R⁹,        wherein the phenyl is optionally substituted with one R¹⁰        substituent;        wherein R⁸ and R⁹ are as defined herein;-   R⁸ and R⁹ are each independently    -   1) H, or    -   2) C₁-C₆ alkyl,        wherein the alkyl is optionally substituted with an aryl; and-   R¹⁰ is    -   1) halogen, or    -   2) OC₁-C₆ alkyl.

In still another subset of the aforesaid compounds,

-   n=1;-   A and A¹ are both CH₂;-   B and B¹ are independently C₁-C₄ alkyl;-   BG is —X-L-X¹; or-   BG is-   X and X¹ are independently selected from-   L is selected from    -   1) —C₁-C₁₀ alkyl-,    -   2) -phenyl-,    -   3) -biphenyl-,    -   4) —CH₂—(C₂-C₄ alkynyl)-CH₂—,    -   5) —CH₂-phenyl-CH₂—,    -   6) —CH₂-biphenyl-CH₂—, or    -   7) —C₁-C₆ alkyl-O—C₁-C₆ alkyl;-   R¹, R¹⁰⁰, R² and R²⁰⁰ are each independently CH₃; and-   Q and Q¹ are both independently selected from:

In one aspect of the present invention, the compounds of the presentinvention may also be represented by Formula 2 in which M1 and M2represent independent BIR binding domains.

wherein n, R¹, R², R¹⁰⁰, R²⁰⁰, A, A¹, Q, Q¹, B, B¹, and BG as definedherein, and the dotted line represents a hypothetical dividing line forcomparing the substituents associated with M1 and M2.

In one subset of compounds of Formula 2, M1 is the same as M2.

In an alternative subset of compounds of Formula 2, M1 is different fromM2.

In still another subset, B is the same as B¹.

In still another subset B is different from B¹.

One skilled in the art will recognize that when M1 and M2 are the same,the R¹, R², R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹³, R¹⁴, m, p, Y, A, Q,and B substituents in M1 have the same meaning as the R¹⁰⁰, R²⁰⁰, R⁴,R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹³, R¹⁴, m, p, Y, A¹, Q¹, and B¹substituents respectively in M2. When M1 and M2 are different, at leastone R¹, R², R¹⁰⁰, R²⁰⁰, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹³, R¹⁴, m,p, Y, A, A¹, Q, Q¹, B, and B¹ substituent is different in either of M1or M2.

Alternatively the substituents in M1 can be defined as R¹, R², R⁴, R⁵,R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹³, R¹⁴, m, p, Y, A, Q, and B, and those inM2 can be defined as R¹⁰⁰, R²⁰⁰, R⁴⁰⁰, R⁵⁰⁰, R⁶⁰⁰, R⁷⁰⁰, R⁸⁰⁰, R⁹⁰⁰,R¹⁰⁰⁰, R¹¹⁰⁰, R¹³⁰⁰, R¹⁴⁰⁰, m¹, p¹, Y¹, A¹, Q¹ and B¹ respectively. Inthe case where M1 and M2 are the same, the R¹, R², R⁴, R⁵, R⁶, R⁷, R⁸,R⁹, R¹⁰, R¹¹, R¹³, R¹⁴, m, p, Y, A, Q, and B substituents in M1 have thesame meanings as R¹⁰⁰, R²⁰⁰, R⁴⁰⁰, R⁵⁰⁰, R⁶⁰⁰, R⁷⁰⁰, R⁸⁰⁰, R⁹⁰⁰, R¹⁰⁰⁰,R¹¹⁰⁰, R¹³⁰⁰, R¹⁴⁰⁰, m¹, p¹, Y¹, A¹, Q¹ and B¹ respectively in M2. Inthe case where M1 and M2 are different, at least one of the aforesaidsubstituents is different.

If any variable, such as R⁶, R⁶⁰⁰, R¹⁰, R¹⁰⁰⁰ and the like, occurs morethan one time in any constituent structure, the definition of thevariable at each occurrence is independent at every other occurrence. Ifa substituent is itself substituted with one or more substituents, it isto be understood that that the one or more substituents may be attachedto the same carbon atom or different carbon atoms. Combinations ofsubstituents and variables defined herein are allowed only if theyproduce chemically stable compounds.

One skilled in the art will understand that substitution patterns andsubstituents on compounds of the present invention may be selected toprovide compounds that are chemically stable and can be readilysynthesized using the chemistry set forth in the examples and chemistrytechniques well known in the art using readily available startingmaterials.

It is to be understood that many substituents or groups described hereinhave functional group equivalents, which means that the group orsubstituent may be replaced by another group or substituent that hassimilar electronic, hybridization or bonding properties.

Definitions

Unless otherwise specified, the following definitions apply:

The singular forms “a”, “an” and “the” include corresponding pluralreferences unless the context clearly dictates otherwise.

As used herein, the term “comprising” is intended to mean that the listof elements following the word “comprising” are required or mandatorybut that other elements are optional and may or may not be present.

As used herein, the term “consisting of” is intended to mean includingand limited to whatever follows the phrase “consisting of”. Thus thephrase “consisting of” indicates that the listed elements are requiredor mandatory and that no other elements may be present.

As used herein, the term “alkyl” is intended to include both branchedand straight chain saturated aliphatic hydrocarbon groups having thespecified number of carbon atoms, for example, C₁-C₁₀ as in C₁-C₁₀ alkylis defined as including groups having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10carbons in a linear or branched arrangement, and C₁-C₆ as in C₁-C₆-alkylis defined as including groups having 1, 2, 3, 4, 5 or 6 carbons in alinear or branched arrangement, and C₁-C₄ as in C₁-C₄ alkyl is definedas including groups having 1, 2, 3, or 4 carbons in a linear or branchedarrangement. Examples of C₁-C₆-alkyl and C₁-C₄ alkyl as defined aboveinclude, but are not limited to, methyl, ethyl, n-propyl, i-propyl,n-butyl, t-butyl, i-butyl, pentyl and hexyl.

As used herein, the term, “alkenyl” is intended to mean unsaturatedstraight or branched chain hydrocarbon groups having the specifiednumber of carbon atoms therein, and in which at least two of the carbonatoms are bonded to each other by a double bond, and having either E orZ regeochemistry and combinations thereof. For example, C₂-C₆ as inC₂-C₆ alkenyl is defined as including groups having 1, 2, 3, 4, 5, or 6carbons in a linear or branched arrangement, at least two of the carbonatoms being bonded together by a double bond. Examples of C₂-C₆ alkenylinclude ethenyl (vinyl), 1-propenyl, 2-propenyl, 1-butenyl and the like.

As used herein, the term “alkynyl” is intended to mean unsaturated,straight chain hydrocarbon groups having the specified number of carbonatoms therein and in which at least two carbon atoms are bonded togetherby a triple bond. For example C₂-C₄ as in C₂-C₄ alkynyl is defined asincluding groups having 2, 3, or 4 carbon atoms in a chain, at least twoof the carbon atoms being bonded together by a triple bond. Examples ofsuch alkynyls include ethynyl, 1-propynyl, 2-propynyl and the like.

As used herein, the term “cycloalkyl” is intended to mean a monocyclicsaturated aliphatic hydrocarbon group having the specified number ofcarbon atoms therein, for example, C₃-C₇ as in C₃-C₇ cycloalkyl isdefined as including groups having 3, 4, 5, 6, or 7 carbons in amonocyclic arrangement. Examples of C₃-C₇ cycloalkyl as defined aboveinclude, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl,cyclohexyl and cycloheptyl.

As used herein, the term “cycloalkenyl” is intended to mean a monocyclicsaturated aliphatic hydrocarbon group having the specified number ofcarbon atoms therein, for example, C₃-C₇ as in C₃-C₇ cycloalkenyl isdefined as including groups having 3, 4, 5, 6, or 7 carbons in amonocyclic arrangement. Examples of C₃-C₇ cycloalkenyl as defined aboveinclude, but are not limited to, cyclopentenyl, and cyclohexenyl.

As used herein, the term “halo” or “halogen” is intended to meanfluorine, chlorine, bromine and iodine.

As used herein, the term “haloalkyl” is intended to mean an alkyl asdefined above, in which each hydrogen atom may be successively replacedby a halogen atom. Examples of haloalkyls include, but are not limitedto, CH₂F, CHF₂ and CF₃.

As used herein, the term “aryl”, either alone or in combination withanother radical, means a carbocyclic aromatic monocyclic groupcontaining 6 carbon atoms which may be further fused to a second 5- or6-membered carbocyclic group which may be aromatic, saturated orunsaturated. Aryl includes, but is not limited to, phenyl, indanyl,1-naphthyl, 2-naphthyl and tetrahydronaphthyl. The fused aryls may beconnected to another group either at a suitable position on thecycloalkyl ring or the aromatic ring. For example:

Arrowed lines drawn from the ring system indicate that the bond may beattached to any of the suitable ring atoms.

As used herein, the term “biphenyl” is intended to mean two phenylgroups bonded together at any one of the available sites on the phenylring. The biphenyl may be covalently bonded to other groups from anyavailable position on the phenyl rings. For example:

As used herein, the term “heteroaryl” is intended to mean a monocyclicor bicyclic ring system of up to ten atoms, wherein at least one ring isaromatic, and contains from 1 to 4 hetero atoms selected from the groupconsisting of O, N, and S. The heteroaryl substituent may be attachedeither via a ring carbon atom or one of the heteroatoms. Examples ofheteroaryl groups include, but are not limited to thienyl,benzimidazolyl, benzo[b]thienyl, furyl, benzofuranyl, pyranyl,isobenzofuranyl, chromenyl, xanthenyl, 2H-pyrrolyl, pyrrolyl,imidazolyl, pyrazolyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl,indolizinyl, isoindolyl, 3H-indolyl, indolyl, indazolyl, purinyl,4H-quinolizinyl, isoquinolyl, quinolyl, phthalazinyl, napthyridinyl,quinoxalinyl, quinazolinyl, cinnolinyl, pteridinyl, isothiazolyl,isochromanyl, chromanyl, isoxazolyl, furazanyl, indolinyl, isoindolinyl,thiazolo[4,5-b]-pyridine, and fluoroscene derivatives such as:

As used herein, the term “heterocycle”, “heterocyclic” or “heterocyclyl”is intended to mean a 5, 6, or 7 membered non-aromatic ring systemcontaining from 1 to 4 heteroatoms selected from the group consisting ofO, N and S. Examples of heterocycles include, but are not limited topyrrolidinyl, tetrahydrofuranyl, piperidyl, pyrrolinyl, piperazinyl,imidazolidinyl, morpholinyl, imidazolinyl, pyrazolidinyl, pyrazolinyl,and

As used herein, the term “heterobicycle” either alone or in combinationwith another radical, is intended to mean a heterocycle as defined abovefused to another cycle, be it a heterocycle, an aryl or any other cycledefined herein. Examples of such heterobicycles include, but are notlimited to, coumarin, benzo[d][1,3]dioxole,2,3-dihydrobenzo[b][1,4]dioxine and3,4-dihydro-2H-benzo[b][1,4]dioepine.

Examples of

wherein G is a 5, 6 or 7 membered ring which optionally incorporates oneor more heteroatoms selected from S, N or O and p is 1 or 2, and isoptionally substituted with one or more R¹² substituents, include, butare not limited to:

As used herein, the term “heteroatom” is intended to mean O, S or N.

As used herein, the term “detectable label” is intended to mean a groupthat may be linked to a compound of the present invention to produce aprobe or to an IAP BIR domain, such that when the probe is associatedwith the BIR domain, the label allows either direct or indirectrecognition of the probe so that it may be detected, measured andquantified.

As used herein, the term “affinity tag” is intended to mean a ligand orgroup, which is linked to either a compound of the present invention orto an IAP BIR domain to allow another compound to be extracted from asolution to which the ligand or group is attached.

As used herein, the term “probe” is intended to mean a compound ofFormula I which is labeled with either a detectable label or an affinitytag, and which is capable of binding, either covalently ornon-covalently, to an IAP BIR domain. When, for example, the probe isnon-covalently bound, it may be displaced by a test compound. When, forexample, the probe is bound covalently, it may be used to formcross-linked adducts, which may be quantified and inhibited by a testcompound.

As used herein, the term “optionally substituted with one or moresubstituents” or its equivalent term “optionally substituted with atleast one substituent” is intended to mean that the subsequentlydescribed event of circumstances may or may not occur, and that thedescription includes instances where the event or circumstance occursand instances in which it does not. The definition is intended to meanfrom zero to five substituents.

If the substituents themselves are incompatible with the syntheticmethods of the present invention, the substituent may be protected witha suitable protecting group (PG) that is stable to the reactionconditions used in these methods. The protecting group may be removed ata suitable point in the reaction sequence of the method to provide adesired intermediate or target compound. Suitable protecting groups andthe methods for protecting and de-protecting different substituentsusing such suitable protecting groups are well known to those skilled inthe art; examples of which may be found in T. Greene and P. Wuts,Protecting Groups in Chemical Synthesis (3^(rd) ed.), John Wiley & Sons,NY (1999), which is incorporated herein by reference in its entirety.Examples of protecting groups used throughout include, but are notlimited to Fmoc, Bn, Boc, CBz and COCF₃. In some instances, asubstituent may be specifically selected to be reactive under thereaction conditions used in the methods of this invention. Under thesecircumstances, the reaction conditions convert the selected substituentinto another substituent that is either useful in an intermediatecompound in the methods of this invention or is a desired substituent ina target compound.

Abbreviations for α-amino acids used throughout are as follows: Aminoacid Abbreviation α-Amino butyric acid Abu Alanine Ala Arginine ArgAspartic acid Asp Asparagine Asn Cysteine Cys Glutamic acid GluGlutamine Gln Glycine Gly Isoleucine Ile Histidine His Leucine LeuLysine Lys Methionine Met Phenylalanine Phe Proline Pro Serine SerThreonine Thr Tryptophan Trp Tyrosine Tyr Valine Val

As used herein, the term “residue” when referring to α-amino acids isintended to mean a radical derived from the corresponding α-amino acidby eliminating the hydroxyl of the carboxy group and one hydrogen of theα-amino group. For example, the terms Gln, Ala, Gly, Ile, Arg, Asp, Phe,Ser, Leu, Cys, Asn, and Tyr represent the residues of L-glutamine,L-alanine, glycine, L-isoleucine, L-arginine, L-aspartic acid,L-phenylalanine, L-serine, L-leucine, L-cysteine, L-asparagine, andL-tyrosine, respectively.

As used herein, the term “subject” is intended to mean humans andnon-human mammals such as primates, cats, dogs, swine, cattle, sheep,goats, horses, rabbits, rats, mice and the like.

As used herein, the term “prodrug” is intended to mean a compound thatmay be converted under physiological conditions or by solvolysis to abiologically active compound of the present invention. Thus, the term“prodrug” refers to a precursor of a compound of the invention that ispharmaceutically acceptable. A prodrug may be inactive or displaylimited activity when administered to a subject in need thereof, but isconverted in vivo to an active compound of the present invention.Typically, prodrugs are transformed in vivo to yield the compound of theinvention, for example, by hydrolysis in blood or other organs byenzymatic processing. The prodrug compound often offers advantages ofsolubility, tissue compatibility or delayed release in the subject (see,Bundgard, H., Design of Prodrugs (1985), pp. 7-9, 21-24 (Elsevier,Amsterdam). The definition of prodrug includes any covalently bondedcarriers which release the active compound of the invention in vivo whensuch prodrug is administered to a subject. Prodrugs of a compound of thepresent invention may be prepared by modifying functional groups presentin the compound of the invention in such a way that the modificationsare cleaved, either in routine manipulation or in vivo, to a parentcompound of the invention.

As used herein, the term “pharmaceutically acceptable carrier, diluentor excipient” is intended to mean, without limitation, any adjuvant,carrier, excipient, glidant, sweetening agent, diluent, preservative,dye/colorant, flavor enhancer, surfactant, wetting agent, dispersingagent, suspending agent, stabilizer, isotonic agent, solvent,emulsifier, or encapsulating agent, such as a liposome, cyclodextrins,encapsulating polymeric delivery systems or polyethyleneglycol matrix,which is acceptable for use in the subject, preferably humans.

As used herein, the term “pharmaceutically acceptable salt” is intendedto mean both acid and base addition salts.

As used herein, the term “pharmaceutically acceptable acid additionsalt” is intended to mean those salts which retain the biologicaleffectiveness and properties of the free bases, which are notbiologically or otherwise undesirable, and which are formed withinorganic acids such as hydrochloric acid, hydrobromic acid, sulfuricacid, nitric acid, phosphoric acid and the like, and organic acids suchas acetic acid, trifluoroacetic acid, propionic acid, glycolic acid,pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid,fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid,mandelic acid, methanesulfonic acid, ethanesulfonic acid,p-toluenesulfonic acid, salicylic acid, and the like.

As used herein, the term “pharmaceutically acceptable base additionsalt” is intended to mean those salts which retain the biologicaleffectiveness and properties of the free acids, which are notbiologically or otherwise undesirable. These salts are prepared fromaddition of an inorganic base or an organic base to the free acid. Saltsderived from inorganic bases include, but are not limited to, thesodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc,copper, manganese, aluminum salts and the like. Salts derived fromorganic bases include, but are not limited to, salts of primary,secondary, and tertiary amines, substituted amines including naturallyoccurring substituted amines, cyclic amines and basic ion exchangeresins, such as isopropylamine, trimethylamine, diethylamine,triethylamine, tripropylamine, ethanolamine, 2-dimethylaminoethanol,2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine,caffeine, procaine, hydrabamine, choline, betaine, ethylenediamine,glucosamine, methylglucamine, theobromine, purines, piperazine,piperidine, N-ethylpiperidine, polyamine resins and the like.

As used herein, the term “BIR domain binding” is intended to mean theaction of a compound of the present invention upon an IAP BIR domain,which blocks or diminishes the binding of IAPs to BIR binding proteinsor is involved in displacing BIR binding proteins from an IAP. Examplesof BIR binding proteins include, but are not limited to, caspases andmitochondrially derived BIR binding proteins such as Smac, Omi/WTR2A andthe like.

As used herein, the term “insufficient apoptosis” is intended to mean astate wherein a disease is caused or continues because cells deleteriousto the subject have not apoptosed. This includes, but is not limited to,cancer cells that survive in a subject without treatment, cancer cellsthat survive in a subject during or following anti-cancer treatment, orimmune cells whose action is deleterious to the subject, and includes,neutrophils, monocytes and auto-reactive T-cells.

As used herein, the term “therapeutically effective amount” is intendedto mean an amount of a compound of Formula I which, when administered toa subject is sufficient to effect treatment for a disease-stateassociated with insufficient apoptosis. The amount of the compound ofFormula I will vary depending on the compound, the condition and itsseverity, and the age of the subject to be treated, but can bedetermined routinely by one of ordinary skill in the art having regardto his own knowledge and to this disclosure.

As used herein, the term “treating” or “treatment” is intended to meantreatment of a disease-state associated with insufficient apoptosis, asdisclosed herein, in a subject, and includes: (i) preventing a diseaseor condition associated with insufficient apoptosis from occurring in asubject, in particular, when such mammal is predisposed to the diseaseor condition but has not yet been diagnosed as having it; (ii)inhibiting a disease or condition associated with insufficientapoptosis, i.e., arresting its development; or (iii) relieving a diseaseor condition associated with insufficient apoptosis, i.e., causingregression of the condition.

As used herein, the term “treating cancer” is intended to mean theadministration of a pharmaceutical composition of the present inventionto a subject, preferably a human, which is afflicted with cancer tocause an alleviation of the cancer by killing, inhibiting the growth, orinhibiting the metastasis of the cancer cells.

As used herein, the term “preventing disease” is intended to mean, inthe case of cancer, the post-surgical, post-chemotherapy orpost-radiotherapy administration of a pharmaceutical composition of thepresent invention to a subject, preferably a human, which was afflictedwith cancer to prevent the regrowth of the cancer by killing, inhibitingthe growth, or inhibiting the metastasis of any remaining cancer cells.Also included in this definition is the prevention of prosurvivalconditions that lead to diseases such as asthma, MS and the like.

As used herein, the term “synergistic effect” is intended to mean thatthe effect achieved with the combination of the compounds of the presentinvention and either the chemotherapeutic agents or death receptoragonists of the invention is greater than the effect which is obtainedwith only one of the compounds, agents or agonists, or advantageouslythe effect which is obtained with the combination of the abovecompounds, agents or agonists is greater than the addition of theeffects obtained with each of the compounds, agents or agonists usedseparately. Such synergy enables smaller doses to be given.

As used herein, the term “apoptosis” or “programmed cell death” isintended to mean the regulated process of cell death wherein a dyingcell displays a set of well-characterized biochemical hallmarks thatinclude cell membrane blebbing, cell soma shrinkage, chromatincondensation, and DNA laddering, as well as any caspase-mediated celldeath.

As used herein, the term “BIR domain” or “BIR” are used interchangeablythroughout and are intended to mean a domain which is characterized by anumber of invariant amino acid residue including conserved cysteines andone conserved hisitidine residue within the sequenceCys-(Xaa1)₂Cys-(Xaa1)₁₆His-(Xaa1)₆₋₈Cys. Typically, the amino acidsequence of the consensus sequence is:Xaa1-Xaa1-Xaa1-Arg-Leu-Xaa1-Thr-Phe-Xaa1-Xaa1-Trp-Pro-Xaa2-Xaa1-Xaa1-Xaa2-Xaa2-Xaa1-Xaa1-Xaa1-Xaa1-Leu-Ala-Xaa1-Ala-Gly-Phe-Tyr-Tyr-Xaa1-Gly-Xaa1-Xaa1-Asp-Xaa1-Val-Xaa1-Cys-Phe-Xaa1-Cys-Xaa1-Xaa1-Xaa1-Xaa1-Xaa1-Xaa1-Trp-Xaa1-Xaa1-Xaa1-Asp-Xaa1-Xaa1-Xaa1-Xaa1-Xaa1-His-Xaa-1-Xaa1-Xaa1-Xaa1-Pro-Xaa1-Cys-Xaa1-Phe-Val,wherein Xaa1 is any amino acid and Xaa2 is any amino acid or is absent.Preferably the sequence is substantially identical to one of the BIRdomain sequences provided for XIAP, HIAP1, or HIAP2 herein.

The BIR domain residues are listed below (see Genome Biology (2001)1-10): XIAP HIAP-1 HIAP-2 BIR1 21-93  41-113 24-96 BIR2 159-230 179-250164-235 BIR3 258-330 264-336 250-322 Seq. # P98170 XP-006266 XP-006267

As used herein, the term “ring zinc finger” or “RZF” is intended to meana domain having the amino acid sequence of the consensus sequence:Glu-Xaa1-Xaa1-Xaa1-Xaa1-Xaa1-Xaa-1-Xaa2-Xaa1-Xaa1-Xaa1-Cys-Lys-Xaa3-Cys-Met-Xaa1-Xaa1-Xaa1-Xaa1-Xaa1-Xaa3-Xaa1-Phe-Xaa1-Pro-Cys-Gly-His-Xaa1-Xaa1-Xaa1-Cys-Xaa1-Xaa1-Cys-Ala-Xaa1-Xaa-1-Xaa1-Xaa1-Xaa1-Cys-Pro-Xaa1-Cys,wherein Xaa1 is any amino acid, Xaa2 is Glu or Asp, and Xaa3 is Val orIle.

As used herein, the term “IAP” is intended to mean a polypeptide orprotein, or fragment thereof, encoded by an IAP gene. Examples of IAPsinclude, but are not limited to human or mouse NAIP (Birc 1), HIAP-1(cIAP2, Birc 3), HIAP-2 (cIAP1, Birc 2), XIAP (Birc 4), survivin (Birc5), livin (ML-IAP, Birc 7), ILP-2 (Birc 8) and Apollon/BRUCE (Birc 6)(see for example U.S. Pat. Nos. 6,107,041; 6,133,437; 6,156,535;6,541,457; 6,656,704; 6,689,562; Deveraux and Reed, Genes Dev. 13,239-252, 1999; Kasof and Gomes, J. Biol. Chem., 276, 3238-3246, 2001;Vucic et al., Curr. Biol. 10, 1359-1366, 2000; Ashab et al. FEBS Lett.,495, 56-60, 2001, the contents of which are hereby incorporated byreference).

As used herein, the term “IAP gene” is intended to mean a gene encodinga polypeptide having at least one BIR domain and which is capable ofmodulating (inhibiting or enhancing) apoptosis in a cell or tissue. TheIAP gene is a gene having about 50% or greater nucleotide sequenceidentity to at least one of human or mouse NAIP (Birc 1), HIAP-1 (cIAP2,Birc 3), HIAP-2 (cIAP1, Birc 2), XIAP (Birc 4), survivin (Birc 5), livin(ML-IAP, Birc 7), ILP-2 (Birc 8) and Apollon/BRUCE (Birc 6). The regionof sequence over which identity is measured is a region encoding atleast one BIR domain and a ring zinc finger domain. Mammalian IAP genesinclude nucleotide sequences isolated from any mammalian source.

As used herein, the term “IC₅₀” is intended to mean an amount,concentration or dosage of a particular compound of the presentinvention that achieves a 50% inhibition of a maximal response, such asdisplacement of maximal fluorescent probe binding in an assay thatmeasures such response.

As used herein, the term “EC₅₀” is intended to mean an amount,concentration or dosage of a particular compound of the presentinvention that achieves a 50% inhibition of cell survival.

As used herein, the term “modulate” or “modulating” is intended to meanthe treatment, prevention, suppression, enhancement or induction of afunction or condition using the compounds of the present invention. Forexample, the compounds of the present invention can modulate IAPfunction in a subject, thereby enhancing apoptosis by significantlyreducing, or essentially eliminating the interaction of activatedapoptotic proteins, such as caspase-3, 7 and 9, with the BIR domains ofmammalian IAPs or by inducing the loss of IAP protein in a cell.

As used herein, the term “enhancing apoptosis” is intended to meanincreasing the number of cells that apoptose in a given cell populationeither in vitro or in vivo. Examples of cell populations include, butare not limited to, ovarian cancer cells, colon cancer cells, breastcancer cells, lung cancer cells, pancreatic cancer cells, or T cells andthe like. It will be appreciated that the degree of apoptosisenhancement provided by an apoptosis-enhancing compound of the presentinvention in a given assay will vary, but that one skilled in the artcan determine the statistically significant change in the level ofapoptosis that identifies a compound that enhances apoptosis otherwiselimited by an IAP. Preferably “enhancing apoptosis” means that theincrease in the number of cells undergoing apoptosis is at least 25%,more preferably the increase is 50%, and most preferably the increase isat least one-fold. Preferably the sample monitored is a sample of cellsthat normally undergo insufficient apoptosis (i.e., cancer cells).Methods for detecting the changes in the level of apoptosis (i.e.,enhancement or reduction) are described in the Examples and includemethods that quantitate the fragmentation of DNA, methods thatquantitate the translocation phosphatoylserine from the cytoplasmic tothe extracellular side of the membrane, determination of activation ofthe caspases and methods quantitate the release of cytochrome C and theapoptosis inhibitory factor into the cytoplasm by mitochondria.

As used herein, the term “proliferative disease” or “proliferativedisorder” is intended to mean a disease that is caused by or results ininappropriately high levels of cell division, inappropriately low levelsof apoptosis, or both. For example, cancers such as lymphoma, leukemia,melanoma, ovarian cancer, breast cancer, pancreatic cancer, and lungcancer, and autoimmune disorders are all examples of proliferativediseases.

As used herein, the term “death receptor agonist” is intended to mean anagent capable of stimulating by direct or indirect contact the proapoptotic response mediated by the death-receptors. For example, anagonist TRAIL receptor Antibody would bind to TRAIL receptor (S) andtrigger an apoptotic response. On the other hand, other agent such asinterferon-a could trigger the release of endogenous TRAIL and/or upregulate the TRAIL receptors in such a way that the cell pro-apoptoticresponse is amplified.

The compounds of the present invention, or their pharmaceuticallyacceptable salts may contain one or more asymmetric centers, chiral axesand chiral planes and may thus give rise to enantiomers, diastereomers,and other stereoisomeric forms and may be defined in terms of absolutestereochemistry, such as (R)- or (S)- or, as (D)- or (L)- for aminoacids. The present invention is intended to include all such possibleisomers, as well as, their racemic and optically pure forms. Opticallyactive (+) and (−), (R)- and (S)-, or (D)- and (L)-isomers may beprepared using chiral synthons or chiral reagents, or resolved usingconventional techniques, such as reverse phase HPLC. The racemicmixtures may be prepared and thereafter separated into individualoptical isomers or these optical isomers may be prepared by chiralsynthesis. The enantiomers may be resolved by methods known to thoseskilled in the art, for example by formation of diastereoisomeric saltswhich may then be separated by crystallization, gas-liquid or liquidchromatography, selective reaction of one enantiomer with an enantiomerspecific reagent. It will also be appreciated by those skilled in theart that where the desired enantiomer is converted into another chemicalentity by a separation technique, an additional step is then required toform the desired enantiomeric form. Alternatively specific enantiomersmay be synthesized by asymmetric synthesis using optically activereagents, substrates, catalysts, or solvents or by converting oneenantiomer to another by asymmetric transformation.

Certain compounds of the present invention may exist in Zwitterionicform and the present invention includes Zwitterionic forms of thesecompounds and mixtures thereof.

Utilities

The compounds of the present invention are useful as IAP BIR domainbinding compounds and as such the compounds, compositions and method ofthe present invention include application to the cells or subjectsafflicted with or having a predisposition towards developing aparticular disease state, which is characterized by insufficientapoptosis. Thus, the compounds, compositions and methods of the presentinvention are used to treat cellular proliferative diseases/disorders,which include, but are not limited to, i) cancer, ii) autoimmunedisease, iii) inflammatory disorders, iv) proliferation induced postmedical procedures, including, but not limited to, surgery, angioplasty,and the like.

The compounds of the present invention may also be useful in thetreatment of diseases in which there is a defect in the programmedcell-death or the apoptotic machinery (TRAIL, FAS, apoptosome), such asmultiple sclerosis, asthma, artherosclerosis, inflammation, autoimmunityand the like.

The treatment involves administration to a subject in need thereof acompound of the present invention or a pharmaceutically acceptable saltthereof, or a pharmaceutical composition comprising a pharmaceuticalcarrier and a therapeutically effective amount of a compound of thepresent invention, or a pharmaceutically acceptable salt thereof.

In particular, the compounds, compositions and methods of the presentinvention are useful for the treatment of cancer including solid tumorssuch as skin, breast, brain, lung, testicular carcinomas, and the like.Cancers that may be treated by the compounds, compositions and methodsof the invention include, but are not limited to the following: TissueExample Adrenal gland neuroblastoma Bone osteogenic sarcoma(osteosarcoma), fibrosarcoma, malignant fibrous histiocytoma,chondrosarcoma, Ewing's sarcoma, malignant lymphoma (reticulum cellsarcoma), multiple myeloma, malignant giant cell tumor chordoma,osteochronfroma (osteocartilaginous exostoses), benign chondroma,chondroblastoma, chondromyxofibroma, osteoid osteoma and giant celltumors Cardiac sarcoma (angiosarcoma, fibrosarcoma, rhabdomyosarcoma,liposarcoma), myxoma, rhabdomyoma, fibroma, lipoma and teratomaGastrointestinal esophagus (squamous cell carcinoma, adenocarcinoma,leiomyosarcoma, lymphoma), stomach (carcinoma, lymphoma,leiomyosarcoma), pancreas (ductal adenocarcinoma, insulinoma,glucagonoma, gastrinoma, carcinoid tumors, vipoma), small bowel(adenocarcinoma, lymphoma, carcinoid tumors, Karposi's sarcoma,leiomyoma, hemangioma, lipoma, neurofibroma, fibroma), large bowel(adenocarcinoma, tubular adenoma, villous adenoma, hamartoma, leiomyoma)Genitourinary kidney (adenocarcinoma, Wilm's tumor [nephroblastoma],tract lymphoma, leukemia), bladder and urethra (squamous cell carcinoma,transitional cell carcinoma, adenocarcinoma), prostate (adenocarcinoma,sarcoma), testis (seminoma, teratoma, embryonal carcinoma,teratocarcinoma, choriocarcinoma, sarcoma, interstitial cell carcinoma,fibroma, fibroadenoma, adenomatoid tumors, lipoma) Gynecological uterus(endometrial carcinoma), cervix (cervical carcinoma, pre-tumor cervicaldysplasia), ovaries (ovarian carcinoma [serous cystadenocarcinoma,mucinous cystadenocarcinoma, unclassified carcinoma], granulosa-thecalcell tumors, Sertoli- Leydig cell tumors, dysgerminoma, malignantteratoma), vulva (squamous cell carcinoma, intraepithelial carcinoma,adenocarcinoma, fibrosarcoma, melanoma), vagina (clear cell carcinoma,squamous cell carcinoma, botryoid sarcoma (embryonal rhabdomyosarcoma),fallopian tubes (carcinoma) Hematologic blood (myeloid leukemia [acuteand chronic], acute lymphoblastic leukemia, chronic lymphocyticleukemia, myeloproliferative diseases, multiple myeloma, myelodysplasticsyndrome), Hodgkin's disease, non-Hodgkin's lymphoma [malignantlymphoma] Liver hepatoma (hepatocellular carcinoma), cholangiocarcinoma,hepatoblastoma, angiosarcoma, hepatocellular adenoma, hemangioma Lungbronchogenic carcinoma (squamous cell, undifferentiated small cell,undifferentiated large cell, adenocarcinoma), alveolar (bronchiolar)carcinoma, bronchial adenoma, sarcoma, lymphoma, chondromatoushamartoma, mesothelioma Nervous system skull (osteoma, hemangioma,granuloma, xanthoma, osteitis deformans), meninges (meningioma,meningiosarcoma, gliomatosis), brain (astrocytoma, medulloblastoma,glioma, ependymoma, germinoma [pinealoma], glioblastoma multiform,oligodendroglioma, schwannoma, retinoblastoma, congenital tumors),spinal cord neurofibroma, meningioma, glioma, sarcoma) Skin malignantmelanoma, basal cell carcinoma, squamous cell carcinoma, Karposi'ssarcoma, moles dysplastic nevi, lipoma, angioma, dermatofibroma, keloids

The compounds of the present invention, or their pharmaceuticallyacceptable salts or their prodrugs, may be administered in pure form orin an appropriate pharmaceutical composition, and can be carried out viaany of the accepted modes of Galenic pharmaceutical practice.

The pharmaceutical compositions of the present invention can be preparedby admixing a compound of the present invention with an appropriatepharmaceutically acceptable carrier, diluent or excipient, and may beformulated into preparations in solid, semi-solid, liquid or gaseousforms, such as tablets, capsules, powders, granules, ointments,solutions, suppositories, injections, inhalants, gels, microspheres, andaerosols. Typical routes of administering such pharmaceuticalcompositions include, without limitation, oral, topical, transdermal,inhalation, parenteral (subcutaneous injections, intravenous,intramuscular, intrasternal injection or infusion techniques),sublingual, ocular, rectal, vaginal, and intranasal. Pharmaceuticalcompositions of the present invention are formulated so as to allow theactive ingredients contained therein to be bioavailable uponadministration of the composition to a subject. Compositions that willbe administered to a subject or patient take the form of one or moredosage units, where for example, a tablet may be a single dosage unit,and a container of a compound of the present invention in aerosol formmay hold a plurality of dosage units. Actual methods of preparing suchdosage forms are known, or will be apparent, to those skilled in thisart; for example, see Remington's Pharmaceutical Sciences, 18th Ed.,(Mack Publishing Company, Easton, Pa., 1990). The composition to beadministered will, in any event, contain a therapeutically effectiveamount of a compound of the present invention, or a pharmaceuticallyacceptable salt thereof, for treatment of a disease-state as describedabove.

A pharmaceutical composition of the present invention may be in the formof a solid or liquid. In one aspect, the carrier(s) are particulate, sothat the compositions are, for example, in tablet or powder form. Thecarrier(s) may be liquid, with the compositions being, for example, anoral syrup, injectable liquid or an aerosol, which is useful in, forexample inhalatory administration.

For oral administration, the pharmaceutical composition is preferably ineither solid or liquid form, where semi-solid, semi-liquid, suspensionand gel forms are included within the forms considered herein as eithersolid or liquid.

As a solid composition for oral administration, the pharmaceuticalcomposition may be formulated into a powder, granule, compressed tablet,pill, capsule, chewing gum, wafer or the like form. Such a solidcomposition will typically contain one or more inert diluents or ediblecarriers. In addition, one or more of the following may be present:binders such as carboxymethylcellulose, ethyl cellulose,microcrystalline cellulose, gum tragacanth or gelatin; excipients suchas starch, lactose or dextrins, disintegrating agents such as alginicacid, sodium alginate, Primogel, corn starch and the like; lubricantssuch as magnesium stearate or Sterotex; glidants such as colloidalsilicon dioxide; sweetening agents such as sucrose or saccharin; aflavoring agent such as peppermint, methyl salicylate or orangeflavoring; and a coloring agent.

When the pharmaceutical composition is in the form of a capsule, e.g., agelatin capsule, it may contain, in addition to materials of the abovetype, a liquid carrier such as polyethylene glycol or oil such assoybean or vegetable oil.

The pharmaceutical composition may be in the form of a liquid, e.g., anelixir, syrup, solution, emulsion or suspension. The liquid may be fororal administration or for delivery by injection, as two examples. Whenintended for oral administration, preferred composition contain, inaddition to the present compounds, one or more of a sweetening agent,preservatives, dye/colorant and flavor enhancer. In a compositionintended to be administered by injection, one or more of a surfactant,preservative, wetting agent, dispersing agent, suspending agent, buffer,stabilizer and isotonic agent may be included.

The liquid pharmaceutical compositions of the present invention, whetherthey be solutions, suspensions or other like form, may include one ormore of the following adjuvants: sterile diluents such as water forinjection, saline solution, preferably physiological saline, Ringer'ssolution, isotonic sodium chloride, fixed oils such as synthetic mono ordiglycerides which may serve as the solvent or suspending medium,polyethylene glycols, glycerin, propylene glycol or other solvents;antibacterial agents such as benzyl alcohol or methyl paraben;antioxidants such as ascorbic acid or sodium bisulfite; chelating agentssuch as ethylenediamine tetraacetic acid; buffers such as acetates,citrates or phosphates and agents for the adjustment of tonicity such assodium chloride or dextrose. The parenteral preparation can be enclosedin ampoules, disposable syringes or multiple dose vials made of glass orplastic. An injectable pharmaceutical composition is preferably sterile.

A liquid pharmaceutical composition of the present invention used foreither parenteral or oral administration should contain an amount of acompound of the present invention such that a suitable dosage will beobtained. Typically, this amount is at least 0.01% of a compound of thepresent invention in the composition. When intended for oraladministration, this amount may be varied to be between 0.1 and about70% of the weight of the composition. For parenteral usage, compositionsand preparations according to the present invention are prepared so thata parenteral dosage unit contains between 0.01 to 1% by weight of thecompound of the present invention.

The pharmaceutical composition of the present invention may be used fortopical administration, in which case the carrier may suitably comprisea solution, emulsion, ointment or gel base. The base, for example, maycomprise one or more of the following: petrolatum, lanolin, polyethyleneglycols, bee wax, mineral oil, diluents such as water and alcohol, andemulsifiers and stabilizers. Thickening agents may be present in apharmaceutical composition for topical administration. If intended fortransdermal administration, the composition may include a transdermalpatch or iontophoresis device. Topical formulations may contain aconcentration of the compound of the present invention from about 0.1 toabout 10% w/v (weight per unit volume).

The pharmaceutical composition of the present invention may be used forrectal administration to treat for example, colon cancer, in the form,e.g., of a suppository, which will melt in the rectum and release thedrug. The composition for rectal administration may contain anoleaginous base as a suitable nonirritating excipient. Such basesinclude, without limitation, lanolin, cocoa butter and polyethyleneglycol.

The pharmaceutical composition of the present invention may includevarious materials, which modify the physical form of a solid or liquiddosage unit. For example, the composition may include materials thatform a coating shell around the active ingredients. The materials thatform the coating shell are typically inert, and may be selected from,for example, sugar, shellac, and other enteric coating agents.Alternatively, the active ingredients may be encased in a gelatincapsule.

The pharmaceutical composition of the present invention in solid orliquid form may include an agent that binds to the compound of thepresent invention and thereby assists in the delivery of the compound.Suitable agents that may act in this capacity include, but are notlimited to, a monoclonal or polyclonal antibody, a protein or aliposome.

The pharmaceutical composition of the present invention may consist ofdosage units that can be administered as an aerosol. The term aerosol isused to denote a variety of systems ranging from those of colloidalnature to systems consisting of pressurized packages. Delivery may be bya liquefied or compressed gas or by a suitable pump system thatdispenses the active ingredients. Aerosols of compounds of the presentinvention may be delivered in single phase, bi-phasic, or tri-phasicsystems in order to deliver the active ingredient(s). Delivery of theaerosol includes the necessary container, activators, valves,subcontainers, and the like, which together may form a kit. One skilledin the art, without undue experimentation may determine preferredaerosols.

The pharmaceutical compositions of the present invention may be preparedby methodology well known in the pharmaceutical art. For example, apharmaceutical composition intended to be administered by injection canbe prepared by admixing a compound of the present invention withsterile, distilled water so as to form a solution. A surfactant may beadded to facilitate the formation of a homogeneous solution orsuspension. Surfactants are compounds that non-covalently interact withthe compound of the present invention so as to facilitate dissolution orhomogeneous suspension of the compound in the aqueous delivery system.

The compounds of the present invention, or their pharmaceuticallyacceptable salts, are administered in a therapeutically effectiveamount, which will vary depending upon a variety of factors includingthe activity of the specific compound employed; the metabolic stabilityand length of action of the compound; the age, body weight, generalhealth, sex, and diet of the patient; the mode and time ofadministration; the rate of excretion; the drug combination; theseverity of the particular disorder or condition; and the subjectundergoing therapy. Generally, a therapeutically effective daily dosemay be from about 0.1 mg to about 40 mg/kg of body weight per day ortwice per day of a compound of the present invention, or apharmaceutically acceptable salt thereof.

Combination Therapy

The compounds of the present invention, or pharmaceutically acceptablesalts thereof, may also be administered simultaneously with, prior to,or after administration of one or more of the therapeutic agentsdescribed below. Such combination therapy may include administration ofa single pharmaceutical dosage formulation which contains a compound ofthe present invention and one or more additional agents given below, aswell as administration of the compound of the present invention and eachadditional agent in its own separate pharmaceutical dosage formulation.For example, a compound of the present invention and a chemotherapeuticagent, such as taxol (paclitaxel), taxotere, etoposide, cisplatin,vincristine, vinblastine, and the like, can be administered to thepatient either together in a single dosage composition, or each agentadministered in separate oral dosage formulations or via intravenousinjection. Where separate dosage formulations are used, the compounds ofthe present invention and one or more additional agents can beadministered at essentially the same time, i.e., concurrently, or atseparately staggered times, i.e., sequentially; combination therapy isunderstood to include all these regimens. In addition, these compoundsmay synergize with molecules that may stimulate the death receptorapoptotic pathway through a direct or indirect manner, as for example,the compounds of the present invention may be used in combination withsoluble TRAIL or any agent that can cause an increase in circulatinglevel of TRAIL, such as interferon-alpha, BCG, or though radiation.

Thus, the present invention also encompasses the use of the compounds ofthe present invention in combination with radiation therapy or one ormore additional agents such as those described in WO 03/099211(PCT/US03/15861), which is hereby incorporated by reference.

Examples of such additional agents include, but are not limited to thefollowing:

-   a) an estrogen receptor modulator,-   b) an androgen receptor modulator,-   c) retinoid receptor modulator,-   d) a cytotoxic agent,-   e) an antiproliferative agent,-   f) a prenyl-protein transferase inhibitor,-   g) an HMG-CoA reductase inhibitor,-   h) an HIV protease inhibitor,-   i) a reverse transcriptase inhibitor,-   k) an angiogenesis inhibitor,-   l) a PPAR-.γ agonist,-   m) a PPAR-.δ. agonist,-   n) an inhibitor of inherent multidrug resistance,-   o) an anti-emetic agent,-   p) an agent useful in the treatment of anemia,-   q) agents useful in the treatment of neutropenia,-   r) an immunologic-enhancing drug.-   s) a proteasome inhibitor such as Velcade and MG132    (7-Leu-Leu-aldehyde) (see He at al. in Oncogene (2004) 23,    2554-2558);-   t) an HDAC inhibitor, such as sodium butyrate, phenyl butyrate,    hydroamic acids, cyclin tetrapeptide and the like (see Rosato et    al., Molecular Cancer Therapeutics 2003, 1273-1284);-   u) an inhibitor of the chemotrypsin-like activity in the proteasome;-   v) E3 ligase inhibitors;-   w) a modulator of the immune system such as, but not limited to,    interferon-alpha or BCG that can induce the release of cytokines,    such as the interleukins, TNF, or induce release of death receptor    ligands such as TRAIL;-   x) a modulator of death receptors TRAIL and TRAIL agonists such as    the humanized antibodies HGS-ETR1 and HGS-ETR2; and    or in combination or sequentially with radiation therapy, so as to    treat the cancer.

Additional combinations may also include agents which reduce thetoxicity of the aforesaid agents, such as hepatic toxicity, neuronaltoxicity, nephrotoxicity and the like.

In one example, co-administration of one of the compounds of Formula Iof the present invention with a death receptor agonist such as TRAIL,such as a small molecule or an antibody that mimics TRAIL may cause anadvantageous synergistic effect. Moreover, the compounds of the presentinvention may be used in combination with any compounds that cause anincrease in circulating levels of TRAIL.

Vinca Alkaloids and Related Compounds

Vinca alkaloids that can be used in combination with the nucleobaseoligomers of the invention to treat cancer and other neoplasms includevincristine, vinblastine, vindesine, vinflunine, vinorelbine, andanhydrovinblastine.

Dolastatins are oligopeptides that primarily interfere with tubulin atthe vinca alkaloid binding domain. These compounds can also be used incombination with the compounds of the invention to treat cancer andother neoplasms. Dolastatins include dolastatin-10 (NCS 376128),dolastatin-15, ILX651, TZT-1027, symplostatin 1, symplostatin 3, andLU103793 (cemadotin).

Cryptophycins (e.g., cryptophycin 1 and cryptophycin 52 (LY355703)) bindtubulin within the vinca alkaloid-binding domain and induce G2/M arrestand apoptosis. Any of these compounds can be used in combination withthe compounds of the invention to treat cancer and other neoplasms.

Other microtubule disrupting compounds that can be used in conjunctionwith the compounds of the invention to treat cancer and other neoplasmsare described in U.S. Pat. Nos. 6,458,765; 6,433,187; 6,323,315;6,258,841; 6,143,721; 6,127,377; 6,103,698; 6,023,626; 5,985,837;5,965,537; 5,955,423; 5,952,298; 5,939,527; 5,886,025; 5,831,002;5,741,892; 5,665,860; 5,654,399; 5,635,483; 5,599,902; 5,530,097;5,521,284; 5,504,191; 4,879,278; and 4,816,444, and U.S. patentapplication Publication Nos. 2003/0153505 A1; 2003/0083263 A1; and2003/0055002 A1, each of which is hereby incorporated by reference.

Taxanes and Other Microtubule Stabilizing Compounds

Taxanes such as paclitaxel, doxetaxel, RPR 109881A, SB-T-1213,SB-T-1250, SB-T-101187, BMS-275183, BRT 216, DJ-927, MAC-321, IDN5109,and IDN5390 can be used in combination with the compounds of theinvention to treat cancer and other neoplasms. Taxane analogs (e.g.,BMS-184476, BMS-188797) and functionally related non-taxanes (e.g.,epothilones (e.g., epothilone A, epothilone B (EP0906), deoxyepothiloneB, and epothilone B lactam (BMS-247550)), eleutherobin, discodermolide,2-epi-discodermolide, 2-des-methyidiscodermolide,5-hydroxymethyldiscodermolide, 19-des-aminocarbonyldiscodermolide,9(13)-cyclodiscodermolide, and laulimalide) can also be used in themethods and compositions of the invention.

Other microtubule stabilizing compounds that can be used in combinationwith the compounds of the invention to treat cancer and other neoplasmsare described in U.S. Pat. Nos. 6,624,317; 6,610,736; 6,605,599;6,589,968; 6,583,290; 6,576,658; 6,515,017; 6,531,497; 6,500,858;6,498,257; 6,495,594; 6,489,314; 6,458,976; 6,441,186; 6,441,025;6,414,015; 6,387,927; 6,380,395; 6,380,394; 6,362,217; 6,359,140;6,306,893; 6,302,838; 6,300,355; 6,291,690; 6,291,684; 6,268,381;6,262,107; 6,262,094; 6,147,234; 6,136,808; 6,127,406; 6,100,411;6,096,909; 6,025,385; 6,011,056; 5,965,718; 5,955,489; 5,919,815;5,912,263; 5,840,750; 5,821,263; 5,767,297; 5,728,725; 5,721,268;5,719,177; 5,714,513; 5,587,489; 5,473,057; 5,407,674; 5,250,722;5,010,099; and 4,939,168; and U.S. patent application Publication Nos.2003/0186965 A1; 2003/0176710 A1; 2003/0176473 A1; 2003/0144523 A1;2003/0134883 A1; 2003/0087888 A1; 2003/0060623 A1; 2003/0045711 A1;2003/0023082 A1; 2002/0198256 A1; 2002/0193361 A1; 2002/0188014 A1;2002/0165257 A1; 2002/0156110 A1; 2002/0128471 A1; 2002/0045609 A1;2002/0022651 A1; 2002/0016356 A1; 2002/0002292 A1, each of which ishereby incorporated by reference.

Other chemotherapeutic agents that may be administered with a compoundof the present invention are listed in the following Table: Alkylatingcyclophosphamide mechlorethamine agents lomustine thiotepa busulfanstreptozocin procarbazine chlorambucil ifosfamide temozolomidealtretamine dacarbazine melphalan semustine estramustine phosphatecarmustine hexamethylmelamine Platinum agents cisplatin tetraplatincarboplatinum BBR-3464 (Hoffmann-La Roche) oxaliplatin OrmiplatinZD-0473 (AnorMED) SM-11355 (Sumitomo) spiroplatinum iproplatinlobaplatin (Aeterna) AP-5280 (Access) carboxyphthalatoplatinumsatraplatin (Johnson Matthey) Antimetabolites azacytidine6-mercaptopurine tomudex hydroxyurea gemcitabine 6-thioguaninetrimetrexate decitabine (SuperGen) capecitabine cytarabindeoxycoformycin clofarabine (Bioenvision) 5-fluorouracil 2-fluorodeoxyfludarabine cytidine floxuridine irofulven (MGI Pharma) methotrexatepentostatin DMDC (Hoffmann-La Roche) 2-chlorodeoxyadenosine idatrexateraltitrexed ethynylcytidine (Taiho) Topoisomerase amsacrine TAS-103(Taiho) inhibitors rubitecan (SuperGen) Topotecan epirubicinelsamitrucin (Spectrum) dexrazoxanet exatecan mesylate (Daiichi)(TopoTarget) etoposide J-107088 (Merck & Co) quinamed (ChemGenex)pixantrone (Novuspharma) teniposide or mitoxantrone BNP-1350(BioNumerik) gimatecan (Sigma-Tau) rebeccamycin analogue (Exelixis)irinotecan (CPT-11) CKD-602 (Chong Kun Dang) diflomotecan(Beaufour-Ipsen) BBR-3576 (Novuspharma) 7-ethyl-10-hydroxy-camptothecinKW-2170 (Kyowa Hakko) Antitumor dactinomycin (actinomycin D) bleomycinicacid antibiotics amonafide idarubicin doxorubicin (adriamycin) bleomycinA azonafide rubidazone deoxyrubicin bleomycin B anthrapyrazoleplicamycinp valrubicin mitomycin C oxantrazole porfiromycin daunorubicin(daunomycin) MEN-10755 (Menarini) losoxantronecyanomorpholinodoxorubicin epirubicin GPX-100 (Gem Pharmaceuticals)bleomycin sulfate (blenoxane) mitoxantrone (novantrone) therarubicinAntimitotic paclitaxel RPR 109881 A (Aventis) agents SB 408075(GlaxoSmithKline) ZD 6126 (AstraZeneca) docetaxel TXD 258 (Aventis)E7010 (Abbott) PEG-paclitaxel (Enzon) Colchicines epothilone B(Novartis) PG-TXL (Cell Therapeutics) AZ10992 (Asahi) vinblastine T900607 (Tularik) IDN 5109 (Bayer) IDN-5109 (Indena) Vincristine T 138067(Tularik) A 105972 (Abbott) AVLB (Prescient NeuroPharma) Vinorelbinecryptophycin 52 (Eli Lilly) A 204197 (Abbott) azaepothilone B (BMS)Vindesine vinflunine (Fabre) LU 223651 (BASF) BNP-7787 (BioNumerik)dolastatin 10 (NCI) auristatin PE (Teikoku Hormone) D 24851 (ASTAMedica)CA-4 prodrug (OXiGENE) rhizoxin (Fujisawa) BMS 247550 (BMS) ER-86526(Eisai) dolastatin-10 (NIH) mivobulin (Warner-Lambert) BMS 184476(BMS)combretastatin A4 (BMS) CA-4 (OXiGENE) cemadotin (BASF) BMS 188797 (BMS)isohomohalichondrin-B (PharmaMar) taxoprexin (Protarga) AromataseAminoglutethimide anastrazole inhibitors Exemestane YM-511 (Yamanouchi)Letrozole formestane atamestane (BioMedicines) Thymidylate pemetrexed(Eli Lilly) ZD-9331 (BTG) synthase nolatrexed (Eximias) CoFactor ™(BioKeys) inhibitors DNA trabectedin (PharmaMar) albumin + 32P (IsotopeSolutions) antagonists mafosfamide (Baxter International) O6 benzylguanine (Paligent) glufosfamide (Baxter International) thymectacin(NewBiotics) edotreotide apaziquone (Spectrum (Novartis)Pharmaceuticals) Farnesyltransferase arglabin (NuOncology Labs) perillylalcohol (DOR BioPharma) inhibitors tipifarnib (Johnson & Johnson)BAY-43-9006 (Bayer) lonafarnib (Schering-Plough) Pump inhibitors CBT-1(CBA Pharma) tariquidar (Xenova) zosuquidar trihydrochloride (Elibiricodar dicitrate (Vertex) Lilly) MS-209 (Schering AG) Histonetacedinaline (Pfizer) depsipeptide (Fujisawa) acetyltransferasepivaloyloxymethyl butyrate (Titan) MS-275 (Schering AG) inhibitors SAHA(Aton Pharma) Metalloproteinase Neovastat (Aeterna Laboratories)marimastat (British Biotech) BMS- inhibitors CMT-3 (CollaGenex) 275291(Celltech) Ribonucleoside gallium maltolate (Titan) triapine (Vion)reductase tezacitabine (Aventis) didox (Molecules for Health) inhibitorsTNF alpha virulizin (Lorus Therapeutics) CDC-394 (Celgene)agonists/antagonists revimid (Celgene) Endothelin A atrasentan (Abbott)ZD-4054 (AstraZeneca) receptor YM-598 (Yamanouchi) antagonist Retinoicacid fenretinide (Johnson & Johnson) LGD-1550 (Ligand) receptor agonistsalitretinoin (Ligand) Immuno- Interferon norelin (Biostar) modulatorsdexosome therapy (Anosys) IRX-2 (Immuno-Rx) oncophage (Antigenics)BLP-25 (Biomira) pentrix (Australian Cancer PEP-005 (Peplin Biotech)Technology) MGV (Progenics) GMK (Progenics) synchrovax vaccines (CTLImmuno) ISF-154 (Tragen) beta.-alethine (Dovetail) adenocarcinomavaccine (Biomira) melanoma vaccine (CTL Immuno) cancer vaccine(Intercell) CLL therapy (Vasogen) CTP-37 (A VI BioPharma) p21 RASvaccine (GemVax) Hormonal and estrogens bicalutamide antihormonalPrednisone testosterone propionate; agents conjugated estrogensfluoxymesterone methylprednisolone flutamide ethinyl estradiolmethyltestosterone prednisolone octreotide chlortrianisendiethylstilbestrol aminoglutethimide nilutamide idenestrol megestrolleuprolide mitotane tamoxifen hydroxyprogesterone caproate P-04(Novogen) goserelin Toremofine medroxyprogesterone 2-methoxyestradiol(EntreMed) leuporelin dexamethasone testosterone arzoxifene (Eli Lilly)Photodynamic talaporfin (Light Sciences) motexafin agentsPd-bacteriopheophorbide (Yeda) gadolinium (Pharmacyclics) Theralux(Theratechnologies) hypericin lutetium texaphyrin (Pharmacyclics)Tyrosine Kinase imatinib (Novartis) C225 (ImClone) Inhibitors kahalide F(PharmaMar) ZD4190 (AstraZeneca) leflunomide (Sugen/Pharmacia) rhu-Mab(Genentech) CEP-701 (Cephalon) ZD6474 (AstraZeneca) ZD1839 (AstraZeneca)MDX-H210 (Medarex) CEP-751 (Cephalon) vatalanib (Novartis) erlotinib(Oncogene Science) 2C4 (Genentech) MLN518 (Millenium) PKI166 (Novartis)canertinib (Pfizer) MDX-447 (Medarex) PKC412 (Novartis) GW2016(GlaxoSmithKline) squalamine (Genaera) ABX-EGF (Abgenix) phenoxodiol ()EKB-509 (Wyeth) SU5416 (Pharmacia) IMG-1C11 (ImClone) trastuzumab(Genentech) EKB-569 (Wyeth) SU6668 (Pharmacia) Miscellaneous agentsSR-27897 (CCK A inhibitor, Sanofi- gemtuzumab (CD33 antibody, WyethAyerst) Synthelabo) CCI-779 (mTOR kinase inhibitor, Wyeth) BCX-1777 (PNPinhibitor, BioCryst) PG2 (hematopoiesis enhancer, Pharmagenesis)tocladesine (cyclic AMP agonist, Ribapharm) exisulind (PDE V inhibitor,Cell Pathways) ranpirnase (ribonuclease stimulant, Alfacell) Immunol ™(triclosan oral rinse, Endo) alvocidib (CDK inhibitor, Aventis) CP-461(PDE V inhibitor, Cell Pathways) galarubicin (RNA synthesis inhibitor,Dong-A) triacetyluridine (uridine prodrug, Wellstat) CV-247 (COX-2inhibitor, Ivy Medical) AG-2037 (GART inhibitor, Pfizer) tirapazamine(reducing agent, SRI International) SN-4071 (sarcoma agent, SignatureBioScience) P54 (COX-2 inhibitor, Phytopharm) WX-UK1 (plasminogenactivator inhibitor, N-acetylcysteine (reducing agent, Zambon) Wilex)CapCell ™ (CYP450 stimulant, Bavarian TransMID-107 .TM. (immunotoxin, KSNordic) Biomedix) R-flurbiprofen (NF-kappaB inhibitor, Encore) PBI-1402(PMN stimulant, ProMetic GCS-100 (gal3 antagonist, GlycoGenesys)LifeSciences) 3CPA (NF-kappaB inhibitor, Active Biotech) PCK-3145(apoptosis promotor, Procyon) G17DT immunogen (gastrin inhibitor,Aphton) bortezomib (proteasome inhibitor, Millennium) seocalcitol(vitamin D receptor agonist, Leo) doranidazole (apoptosis promotor,Pola) efaproxiral (oxygenator, Allos Therapeutics) SRL-172 (T cellstimulant, SR Pharma) CHS- 131-I-TM-601 (DNA antagonist, 828 (cytotoxicagent, Leo) TransMolecular) TLK-286 (glutathione S transferaseinhibitor, PI-88 (heparanase inhibitor, Progen) Telik) eflornithine (ODCinhibitor, ILEX Oncology) trans-retinoic acid (differentiator, NIH)tesmilifene (histamine antagonist, YM PT-100 (growth factor agonist,Point BioSciences) Therapeutics) minodronic acid (osteoclast inhibitor,MX6 (apoptosis promotor, MAXIA) Yamanouchi) midostaurin (PKC inhibitor,Novartis) histamine (histamine H2 receptor agonist, apomine (apoptosispromotor, ILEX Oncology) Maxim) bryostatin-1 (PKC stimulant, GPCBiotech) indisulam (p53 stimulant, Eisai) urocidin (apoptosis promotor,Bioniche) tiazofurin (IMPDH inhibitor, Ribapharm) CDA-II (apoptosispromotor, Everlife) aplidine (PPT inhibitor, PharmaMar) Ro-31-7453(apoptosis promotor, La Roche) cilengitide (integrin antagonist, MerckKGaA) SDX-101 (apoptosis promotor, Salmedix) rituximab (CD20 antibody,Genentech) brostallicin (apoptosis promotor, Pharmacia) SR-31747 (IL-1antagonist, Sanofi-Synthelabo) ceflatonin (apoptosis promotor,ChemGenex)

Additional combinations may also include agents which reduce thetoxicity of the aforesaid agents, such as hepatic toxicity, neuronaltoxicity, nephprotoxicity and the like.

Screening Assays

The compounds of the present invention may also be used in a method toscreen for other compounds that bind to an IAP BIR domain. Generallyspeaking, to use the compounds of the invention in a method ofidentifying compounds that bind to an IAP BIR domain, the IAP is boundto a support, and a compound of the invention is added to the assay.Alternatively, the compound of the invention may be bound to the supportand the IAP is added.

There are a number of ways in which to determine the binding of acompound of the present invention to the BIR domain. In one way, thecompound of the invention, for example, may be fluorescently orradioactively labeled and binding determined directly. For example, thismay be done by attaching the IAP to a solid support, adding a detectablylabeled compound of the invention, washing off excess reagent, anddetermining whether the amount of the detectable label is that presenton the solid support. Numerous blocking and washing steps may be used,which are known to those skilled in the art.

In some cases, only one of the components is labeled. For example,specific residues in the BIR domain may be labeled. Alternatively, morethan one component may be labeled with different labels; for example,using I¹²⁵ for the BIR domain, and a fluorescent label for the probe.

The compounds of the invention may also be used as competitors to screenfor additional drug candidates or test compounds. As used herein, theterms “drug candidate” or “test compounds” are used interchangeably anddescribe any molecule, for example, protein, oligopeptide, small organicmolecule, polysaccharide, polynucleotide, and the like, to be tested forbioactivity. The compounds may be capable of directly or indirectlyaltering the IAP biological activity.

Drug candidates can include various chemical classes, although typicallythey are small organic molecules having a molecular weight of more than100 and less than about 2,500 Daltons. Candidate agents typicallyinclude functional groups necessary for structural interaction withproteins, for example, hydrogen bonding and lipophilic binding, andtypically include at least an amine, carbonyl, hydroxyl, ether, orcarboxyl group. The drug candidates often include cyclical carbon orheterocyclic structures and/or aromatic or polyaromatic structuressubstituted with one or more functional groups.

Drug candidates can be obtained from any number of sources includinglibraries of synthetic or natural compounds. For example, numerous meansare available for random and directed synthesis of a wide variety oforganic compounds and biomolecules, including expression of randomizedoligonucleotides. Alternatively, libraries of natural compounds in theform of bacterial, fungal, plant and animal extracts are available orreadily produced. Additionally, natural or synthetically producedlibraries and compounds are readily modified through conventionalchemical, physical and biochemical means.

Competitive screening assays may be done by combining an IAP BIR domainand a probe to form a probe:BIR domain complex in a first samplefollowed by adding a test compound from a second sample. The binding ofthe test is determined, and a change, or difference in binding betweenthe two samples indicates the presence of a test compound capable ofbinding to the BIR domain and potentially modulating the IAP's activity.

In one case, the binding of the test compound is determined through theuse of competitive binding assays. In this embodiment, the probe islabeled with an an affinity label such as biotin. Under certaincircumstances, there may be competitive binding between the testcompound and the probe, with the probe displacing the candidate agent.

In one case, the test compound may be labeled. Either the test compound,or a compound of the present invention, or both, is added first to theIAP BIR domain for a time sufficient to allow binding to form a complex.

Formation of the probe:BIR domain complex typically require Incubationsof between 4° C. and 40° C. for between 10 minutes to about 1 hour toallow for high-throughput screening. Any excess of reagents aregenerally removed or washed away. The test compound is then added, andthe presence or absence of the labeled component is followed, toindicate binding to the BIR domain.

In one case, the probe is added first, followed by the test compound.Displacement of the probe is an indication the test compound is bindingto the BIR domain and thus is capable of binding to, and potentiallymodulating, the activity of IAP. Either component can be labeled. Forexample, the presence of probe in the wash solution indicatesdisplacement by the test compound. Alternatively, if the test compoundis labeled, the presence of the probe on the support indicatesdisplacement.

In one case, the test compound may be added first, with incubation andwashing, followed by the probe. The absence of binding by the probe mayindicate the test compound is bound to the BIR domain with a higheraffinity. Thus, if the probe is detected on the support, coupled with alack of test compound binding, may indicate the test compound is capableof binding to the BIR domain.

Modulation is tested by screening for a test compound's ability tomodulate the activity of IAP and includes combining a test compound withan IAP BIR domain, as described above, and determining an alteration inthe biological activity of the IAP. Therefore in this case, the testcompound should both bind to the BIR domain (although this may not benecessary), and alter its biological activity as defined herein.

Positive controls and negative controls may be used in the assays. Allcontrol and test samples are performed multiple times to obtainstatistically significant results. Following incubation, all samples arewashed free of non-specifically bound material and the amount of boundprobe determined. For example, where a radiolabel is employed, thesamples may be counted in a scintillation counter to determine theamount of bound compound.

Typically, the signals that are detected in the assay may includefluorescence, resonance energy transfer, time resolved fluorescence,radioactivity, fluorescence polarization, plasma resonance, orchemiluminescence and the like, depending on the nature of the label.Detectable labels useful in performing screening assays in thisinvention include a fluorescent label such as Fluorescein, Oregon green,dansyl, rhodamine, tetramethyl rhodamine, texas red, Eu³⁺; achemiluminescent label such as luciferase; calorimetric labels;enzymatic markers; or radioisotopes such as tritium, I¹²⁵ and the like

Affinity tags, which may be useful in performing the screening assays ofthe present invention include be biotin, polyhistidine and the like.

Synthesis and Methodology

General methods for the synthesis of the compounds of the presentinvention are shown below and are disclosed merely for the purpose ofillustration and are not meant to be interpreted as limiting theprocesses to make the compounds by any other methods. Those skilled inthe art will readily appreciate that a number of methods are availablefor the preparation of the compounds of the present invention. A numberof intermediate compounds disclosed herein may be synthesized usingsynthetic methods disclosed in previously filed U.S. patent applicationSer. No. 11/434,166, filed May 17, 2006, the entire contents of which ishereby incorporated by reference.

Scheme 1 illustrates the synthesis of a typical synthetic intermediaterepresented by 1(i). Examples of 1(i) represent proline derivatives suchas 1(ii) and 2-(aminomethyl)pyrrolidine derivatives represented byintermediates 1(iii-viii). Proline derivatives of 1(i) may be preparedby the treatment of Boc-Pro-OH with typical peptide coupling agents andan amine, to provide intermediate 1(ii). The 2-(aminomethyl)pyrrolidineintermediate 1(iii) is prepared by the condensation of an amide withN-Boc-prolinal. The resulting amine may be acylated with an acidchloride, anhydride or suitably activated carboxylic acid, such assuccinamidyl esters, HOBt esters and the like, to provide intermediatessuch as 1(iv-vi). The intermediates 1(iv) and 1(v) feature protectinggroups, which may be further removed and functionalized later in thesynthesis. Sulfonylation with a sulfonyl chloride provides 1(vii).Appropriately activated, side chain protected amino acids may be coupledto intermediate 1(iii) using standard peptide coupling agents to provideintermediate 1(viii), the PG can be removed later in the synthesis.

General Procedure for the Preparation of bis-alkynyl Derivatives ofFormula 1g

Scheme 2 illustrates a general procedure for preparing bis-alkynylbridged compounds of formula Ig. PG¹-Thr-OH is deprotonated with NaH andtreated with propargyl bromide to provide the Thr intermediate 2(i).Activation of the carboxylic acid of 2(i) with standard peptide couplingagents and treatment with intermediate 1(i) provides the amideintermediate 2(ii). Peptide coupling of PG²(R¹)N(R²)(H)CCO₂H with 2(ii)is effected by activation of the carboxylic acid of PG²(R¹)N(R²)(H)CCO₂Hwith standard peptide coupling agents, followed by the addition of 2(ii)to provide the fully protected amide 2(iii). The bis-alkynyl bridgingmoiety is prepared by homo-coupling if the alkyne moieties of 2(iii)using an appropriate Cu catalyst, and subsequent deprotection of PG², toprovide compounds of formula Ig.

General Procedure for the Preparation of Compounds of Formula Ih.

Scheme 3 illustrates a general procedure for the preparation ofdi(bromomethyl)benzene derived compounds of Formula I. PG¹-Ser-OH isdeprotonated with NaH and treated with 1,4-di(bromomethyl)benzene toprovide the Ser intermediate 3(i). Activation of the carboxylic acid of3(i) with standard peptide coupling agents and treatment withintermediate 1(i) provides intermediate 3(ii), which is deprotected atPG¹ to provide the amide intermediate 3(iii). Peptide coupling ofPG²(R¹)N(R²)(H)CCO₂H with 3(iii) is effected by activation of thecarboxylic acid of PG²(R¹)N(R²)(H)CCO₂H with standard peptide couplingagents, followed by the addition of 3(iii) to provide the fullyprotected amide, which may be further deprotected at PG² to providecompounds of formula Ih.

General Procedure for the Preparation of Symmetric Amides of Formula I-jand I-k

Scheme 4 depicts a general procedure for the preparation of symmetricamides of Formula 1-f and 1-g. Activation of the carboxylic acid ofPG¹-Om(PG²)-OH with standard peptide coupling agents and treatment withintermediate 1(i), followed by deprotection of PG¹, provides the amideintermediate 4(i). Peptide coupling of PG³(R¹)N(R²)(H)CCO₂H with 4(i) iseffected by activation of the carboxylic acid of PG³(R¹)N(R²)(H)CCO₂Hwith standard peptide coupling agents, followed by the addition of 4(i)to provide the fully protected amide 4(ii). Selective removal of PG²provides the amine intermediate 4(iii). Treatment of 4(iii) with 0.5equiv of an activated alkyl or aromatic diacid, followed by deprotectionof PG³, provides compounds of formula I-j and I-k, respectively.

General Procedure for the Preparation of Compounds of Formula I-l

Scheme 5 illustrates a general procedure for the preparation ofsymmetrical ureas of general formula I-l. Intermediate 4(iii) is treatedwith 0.5 equiv of triphosgene, or a triphosgene equivalent, to provide aprotected urea intermediate 5(i). Removal of PG³ provides compounds ofgeneral Formula I-l.

General Procedure for the Preparation of Symmetrical Esters

Scheme 6 illustrates the preparation of symmetrical esters of generalformula 1-m and I-n. An amino acid derivative displaying a hydroxymoeity on its side chain such as PG¹-Ser(PG²)-OH is activated withstandard peptide coupling reagents and treated with 1(i), and theresulting amide is deprotected at PG¹ to provide the amine intermediate6(i). Activation of the carboxylic acid of PG³(R³)N(H)(R²)CCO₂H usingstandard peptide coupling agents and treatment of the resultingactivated amino acid with 6(i) provides 6(ii). Selective deprotection ofPG² provides the intermediate alcohol 6(iii). Treatment of 6(iii) with0.5 equiv of an activated dicarboxylic acid, and deprotection of PG³,provides compounds of general formula I-m and I-n.

General Procedure for the Preparation of Symmetrical Amides of FormulaI-o

Scheme 7 illustrates the preparation of symmetrical amides of generalformula 1-o. An amino acid derivative displaying a carboxylic acid onits side chain such as PG¹-Glu(PG²)-OH is activated with standardpeptide coupling reagents and treated with 1(i) and the resulting amideis deprotected at PG¹ to provide the amine intermediate 7(ii).Activation of the carboxylic acid of PG³(R³)N(R²)(H)CCO₂H using standardpeptide coupling agents, followed by treatment with 7(i) provides 7(ii).Selective deprotection of PG² provides the intermediate carboxylic acid7(iii). Activation of the carboxylic acid with standard peptide couplingagents and treatment with 0.5 equiv of a diamine provides intermediate7(iv). Deprotection of PG³ provides compounds of general formula I-o.

General Procedure for the Preparation of Compounds of Formula Ii.

Scheme 8 illustrates a general procedure for the preparation ofcompounds of Formula Ii. PG¹-Ser-OH is deprotonated with NaH and treatedwith 2,2′-bis(bromomethyl)-1,1′-biphenyl to provide the Ser intermediate8(i). Activation of the carboxylic acid of 8(i) with standard peptidecoupling agents and treatment with intermediate 1(i) providesintermediate 8(ii), which is deprotected at PG¹ to provide the amideintermediate 8(iii). Peptide coupling of PG²(R¹)N(R²)(H)CCO₂H with3(iii) is effected by activation of the carboxylic acid ofPG²(R¹)N(R²)CHCO₂H with standard peptide coupling agents, followed bythe addition of 3(ii) to provide the fully protected amide, which may befurther deprotected at PG² to provide compounds of formula Ii.

General Procedure for the Preparation of Compounds of Formula Ip

Scheme 9 illustrates a general procedure for the preparation ofglyoxalamides of general formula Ip. Intermediate 4(iii) is treated with0.5 equiv of oxalyl chloride, or an oxalyl chloride equivalent, toprovide a protected urea intermediate 9(i). Removal of PG³ providescompounds of general formula Ip.

General Procedure for the Preparation of Compounds of Formula Iq

Reduction of the triple bonds of Compounds of general formula 1g providecompounds of the general formula 1q. For example, hydrogenation ofcompounds of general formula 1g with H₂ gas in the presence of acatalyst system such as Pd/C provides compounds of general formula 1p.

The above Schemes are applicable to both symmetrical compounds andunsymmetrical compounds of the present invention. The substituents B,B¹, A¹, A, Q, Q¹, R¹, R¹⁰⁰, R², R²⁰⁰, R⁴, R⁵, R¹¹, r and the like are asdefined herein.

EXAMPLES

The following abbreviations are used throughout:

-   Boc: t-butoxycarbonyl;-   CBz: benzyloxycarbonyl;-   DCM: dichloromethane;-   DIPEA: diisopropylethylamine;-   DMAP: 4-(dimethylamino)pyridine;-   DMF: N,N-dimethylformamide;-   DTT: dithiothreitol;-   EDC: 3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride;-   EDTA: ethylenediaminetetracetic acid;-   Fmoc: N-(9-fluorenylmethoxycarbonyl);-   HBTU: O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium    hexafluorophosphate;-   HCl: hydrochloric acid;-   HOAc: acetic acid;-   HOBt: 1-hydroxybenzotriazole;-   HPLC: high performance liquid chromatography;-   LCMS: liquid chromatography-mass spectrometer;-   MeOH: methanol;-   MgSO₄: magnesium sulfate;-   MS: mass spectrum;-   NaHCO₃: sodium hydrogen carbonate;-   Pd/C: palladium on carbon;-   TEA: triethylamine; and-   THF: tetrahydrofuran.    1. Synthesis of Intermediate 1-4b    Step One:    Step a)

To a solution of N-(tert-butoxycarbonyl)-L-prolinal I-1 (6.0 g, 30.1mmol) in methylene chloride was added phenethylamine (3.8 mL, 30.1mmol). After stirring for 1 h at RT sodium cyanoborohydride (12.8 g,60.2 mmol) was added and the reaction mixture was stirred at roomtemperature overnight. Aqueous NaHCO₃ and ethyl acetate were added, theorganic layer was separated, washed with brine, dried over MgSO₄ andconcentrated in vacuo. Purification by flash chromatography providesI-2a as colorless oil. MS (m/z) M+1=305.

Step b)

To a solution of I-2a (6.0 g, 19.7 mmol) in methylene chloride weresequentially added triethylamine (5.5 mL, 39.5 mmol), 4-dimethylaminopyridine (catalytic) and trifluoroacetic anhydride (4.2 mL, 29.6 mmol)and the reaction mixture was stirred for 3 h at room temperature.Aqueous NaHCO₃ and ethyl acetate were added, the organic layer wasseparated, washed with brine, dried over MgSO₄ and concentrated invacuo. Purification by flash chromatography provides I-2b as colorlessoil.

Step c)

A 4 N solution of HCl in 1,4-dioxane (20 mL) was added to I-2b (7.4 g,18.5 mmol) at room temperature and the solution was stirred for 2 h andthen concentrated in vacuum. Crystallization from ether provides I-2c asa white solid. MS (m/z) M+1=301.Step Two

Step a)

To a solution of I-2d (7.2 g, 21.3 mmol) in DMF were sequentially addedDIPEA (19.0 mL, 106 mmol), HOBt (4.24 b, 27.7 mmol) and HBTU (10.5 g,27.7 mmol). After stirring for 5 min I-2c (7.1 g, 27.7 mmol) was addedand the reaction mixture was stirred overnight at room temperature.Water and ethyl acetate were added, the organic layer was separated,washed with 10% citric acid, aqueous NaHCO₃ and brine, dried over MgSO₄and concentrated in vacuo. Purification by flash chromatography providesI-3a compound as a white solid.

Step b)

A 4 N solution of HCl in 1,4-dioxane (15 mL) was added to I-3a (10.7 g,18.0 mmol) at room temperature and the solution was stirred for 2 h andthen concentrated in vacuo. Crystallization from ether provides I-3b asa white solid. MS (m/z) M+1=440.Step Three

Step a)

To a solution of I-3b (8.9 g, 18.7 mmol) in DMF were sequentially addedDIPEA (16.7 mL, 93.6 mmol), HOBt (3.7 g, 24.3 mmol), HBTU (9.2 g, 24.3mmol). After stirring for 5 min BOC-NMeAlaOH (4.9 g, 24.3 mmol) wasadded and the reaction mixture was stirred overnight at roomtemperature. Water and ethyl acetate were added, the organic layer wasseparated, washed with 10% citric acid, aqueous NaHCO₃ and brine, driedover MgSO₄ and concentrated in vacuo. Purification by flashchromatography provides I-4a as a white solid.

Step b)

To a solution of I-4a (8.7 g, 13.4 mmol) in THF cooled to 0° C. wasadded 2 N LiOH (20 mL) and the reaction was stirred overnight at roomtemperature. PH was adjusted to 6 with 10% citric acid and ethyl acetatewas added, the organic layer was separated, washed with brine dried overMgSO₄ and concentrated in vacuum. Purification by flash chromatographyprovides I-4b as a white solid. MS (m/z) M+1=625.Synthesis of I-2d

To a suspension of NaH (4.56 g, 114.04 mmol) in dry DMF (100 mL) cooledto 0° C. was added portion wise N-Boc-L-threonine (10.00 g, 45.62 mmol).After stirring for 10 min propargyl bromide (10 mL) was slowly added andthe reaction was stirred for 1 hr at 0° C. Water (500 mL) and ethylacetate (100 mL) were added, the organic layer was separated, theaqueous layer was acidified to pH=5 with 10% citric acid and extractedtwice with ethyl acetate. The combined organic extracts were washed withbrine, dried over MgSO₄ and concentrated in vacuo. Purification by flashchromatography provides I-2d as a colorless oil.

2. Synthesis of Compound 4

Step One

Step a)

To a solution of I-4b (600 mg, 1.1 mmol) in THF were sequentially addedDIPEA (240 uL, 2.3 mmol) and benzenesulfonyl chloride (160 uL, 2.2mmol). The reaction was stirred for 1 h at room temperature. Water andethyl acetate were added, the organic layer was separated, washed with10% citric acid and brine, dried over MgSO₄ and concentrated in vacuum.Purification by flash chromatography provides 2-1 as a white solid.Step Two

Step a)

To a solution of 2-1 (400 mg, 0.6 mmol) in dry acetone were sequentiallyadded tetramethylethylenediamine (180 uL, 1.2 mmol) and copper (I)chloride (118 mg, 1.2 mmol). The reaction was stirred overnight at roomtemperature and solvent was removed in vacuo. Water and ethyl acetatewere added, the organic layer was separated, washed with 10% citricacid, aqueous NaHCO₃ and brine, dried over MgSO₄ and concentrated invacuum. Purification by flash chromatography provides 2-1a as a whitesolid.

Step b)

A 4 N solution of HCl in dioxane (3 mL) was added to 2-1a (542 mg, 0.47mmol) at 0° C. The solution was stirred for 2 h and then concentrated invacuo. Crystallization from ether provides compound 4-2HCl as a whitesolid. MS (m/z) M+1=1136.3. Synthesis of Compound 2

Step One

To a solution of I-4b (900 mg, 1.7 mmol) in DMF were sequentially addedDIPEA (1.5 mL, 8.5 mmol), HBTU (841 mg, 2.2 mmol) and HOBt (340 mg, 2.2mmol). After stirring for 5 min Boc-D-Tyr(Me)-OH (655 mg, 2.2 mmol) wasadded and the reaction mixture was stirred overnight at roomtemperature. Water and ethyl acetate were added, the organic layer wasseparated, washed with 10% citric acid, aqueous NaHCO₃ and brine, driedover MgSO₄ and concentrated in vacuo. Purification by flashchromatography provides 3-1 as a white solid.Step Two

Step a)

To a solution of 3-1 (225 mg, 0.3 mmol) in dry acetone were sequentiallyadded tetramethylethylenediamine (85 uL, 0.5 mmol) and copper (I)chloride (54 mg, 0.5 mmol) were added and the reaction was stirredovernight at room temperature and solvent was removed in vacuum, Waterand ethyl acetate were added, the organic layer was separated, washedwith 10% citric acid, aqueous NaHCO₃ and brine, dried over MgSO₄ andconcentrated in vacuo. Purification by flash chromatography provides3-1a as a white solid.

Step b)

A 4 N solution of HCl in 1,4-dioxane (2 mL) was added to 3-1a (150 mg,0.1 mmol) at 0° C. and the solution was stirred for 2 h and thenconcentrated in vacuum. Crystallization from diethyl ether yieldedcompound 2•2HCl as a white solid. MS (m/z) M+1=1210.

4. Synthesis of Compound 11

Step One

To a suspension of NaH (1.46 g, 36.5 mmol) in DMF cooled to 0° C. wasadded BOC-Ser-OH 4-1 (3.0 g, 14.6 mmol) and after stirring for 15 minα,α′-Dibromo-p-xylene (2.3 g, 8.7 mmol) was added. The reaction was thenstirred for 1 h at 0° C. and 15 min at RT. Water was added and PH wasacidified to pH 5 with 1N HCl. Ethyl acetate was added, the organiclayer was separated, washed with brine, dried over MgSO₄ andconcentrated in vacuo. Purification by flash chromatography provides4-1a as a white solid.Step Two

Step a)

To a solution of 4-1a (1.6 g, 3.1 mmol) in DMF were sequentially addedDIPEA (1.3 mL, 7.5 mmol), HOBt (1.2 g, 7.8 mmol) and HBTU (2.9 g, 7.8mmol). After stirring for 5 min 1-2c (1.7 g, 5.7 mmol) was added and thereaction mixture was stirred overnight at room temperature. Water andethyl acetate were added, the organic layer was separated, washed with10% citric acid, aqueous NaHCO₃ and brine, dried over MgSO₄ andconcentrated in vacuo. Purification by flash chromatography provides4-1b as a white solid.

Step b)

A 4 N solution of HCl in 1,4-dioxane (5 mL) was added to 4-1b (1.4 g,1.3 mmol) at room temperature and the solution was stirred for 2 h andthen concentrated in vacuo. Crystallization from ether provides 4-1c asa white solid. MS (m/z) M+1=877.Step Three

Step a)

To a solution of 4-1c (550 mg, 0.6 mmol) in DMF were sequentially addedDIPEA (550 uL, 3.1 mmol), HBTU (611 mg, 1.6 mmol) and HOBt (246 mg, 1.6mmol). After stirring for 5 min BOC-NMe-AlaOH (327 mg, 1.6 mmol) wasadded and the reaction mixture was stirred overnight at roomtemperature. Water and ethyl acetate were added, the organic layer wasseparated, washed with 10% citric acid, aqueous NaHCO₃ and brine, driedover MgSO₄ and concentrated in vacuo. Purification by flashchromatography provides 4-1d as a white solid.

Step b)

A 4 N solution of HCl in 1,4-dioxane (5 mL) was added to 4-1d (520 mg,0.4 mmol) at room temperature and the solution was stirred for 2 h andthen concentrated in vacuum. Crystallization from ether providescompound 11•2HCl as a white solid. MS (m/z) M+1=1048.

5. Synthesis of Compound 18

Step One

Step a)

To a solution of Boc-Glu(OBn)-OH (5.55 g, 16.4 mmol) in DMF weresequentially added DIPEA (12.5 mL, 71.8 mmol), HOBt (3.86 g, 28.6 mmol)and HBTU (5.43 g, 14.3 mmol). After stirring for 5 min I-2c (3.04 g, 9.0mmol) was added and the reaction mixture was stirred overnight at roomtemperature. Water and ethyl acetate were added, the organic layer wasseparated, washed with 10% citric acid, aqueous NaHCO₃ and brine, driedover MgSO₄ and concentrated in vacuo. Purification by flashchromatography provides 5-1a as a white solid.

Step b)

A 4 N solution of HCl in 1,4-dioxane (20 mL) was added to 5-1a (5.2 g,8.4 mmol) at room temperature and the solution was stirred for 2 h andthen concentrated in vacuum. Crystallization from ether provides 5-1b asa white solid.Step Two

Step a)

To a solution of Boc-NMe-Ala-OH (2.1 g, 10.4 mmol) in DMF weresequentially added DIPEA (10.5 mL, 60.3 mmol), HBTU (3.0 g, 9.3 mmol)and HOBt (2.0 g, 15.3 mmol). After stirring for 5 min 5-1b (4.7 g, 8.4mmol) was added and the reaction mixture was stirred overnight at roomtemperature. Water and ethyl acetate were added, the organic layer wasseparated, washed with 10% citric acid, aqueous NaHCO₃ and brine, driedover MgSO₄ and concentrated in vacuo. Purification by flashchromatography provides 5-1c as a white solid.

Step b)

A suspension of 5-1c (1.9 g, 2.8 mmol) and 10% Pd/C (196 mg) was stirredfor 3 hrs under hydrogen atmosphere. The reaction was filtered throughcelite and filtrate concentrated in vacuo. Purification by flashchromatography provides 5-1d as a white solid.Step Three

Step a)

To a solution of 5-1d (101 mg, 0.16 mmol) in DMF were sequentially addedDIPEA (200 uL, 1.1 mmol), HBTU (56 mg, 0.14 mmol) and HOBt (24 mg, 0.18mmol). After stirring for 5 min ethylenediamine (3.7 mg, 0.06 mmol) wasadded and the reaction mixture was stirred overnight at roomtemperature. Water and ethyl acetate were added, the organic layer wasseparated, washed with 10% citric acid, aqueous NaHCO₃ and brine, driedover MgSO₄ and concentrated in vacu. Purification by flashchromatography provides 5-1e as a white solid.

Step b)

A 4 N solution of HCl in 1,4-dioxane (5 mL) was added to 5-1e (75 mg,0.06 mmol) at room temperature and the solution was stirred for 2 h andthen concentrated in vacuum. Crystallization from ether providedcompound 18•HCl as a white solid. MS (m/z) (M+2)/2=527.3.

6. Synthesis of Compound 15

Step One

Step a)

Boc-L-proline (9.36 g, 43.5 mmol), HOBt (8.0 g, 52.2 mmol), EDC (10 g,52.2 mmol) and DIPEA (30 mL, 174 mmol) were dissolved in drydichloromethane (200 mL) under N₂ and stirred for 10 min at roomtemperature. 1,2,3,4-R-Tetrahydronaphtylamine (6.72 g, 45.6 mmol) wasthen added and the solution was left to stir for 24 h at RT. Thecontents were then added to a separatory funnel along with EtOAc andwashed with 10% citric acid (2×), saturated NaHCO₃ (2×) and brine. Theorganic layer was collected, dried and concentrated under reducedpressure to provide 6-1a.

Step b)

The product of step a) was treated with 50% CH₂Cl₂/TFA (50 mL) for 1 hrat room temperature. Volatiles were removed in vacuo. to provide 6-1b asthe TFA salt. MS (m/z) M+1=245.Step Two

Step a)

Z-Orn(Boc)OH (2.63 g, 7.2 mmol), HOBt (1.19 g, 7.8 mmol), HBTU (2.96 g,7.8 mmol) and DIPEA (4.6 mL, 26 mmol) were dissolved in dry DMF (12 ml)under N₂ and stirred for 10 min at room temperature. Intermediate 6-1b(3.0 g, 6.5 mmol) was then added and the solution was left to stir for24 h at room temperature. The contents were then added to a separatoryfunnel along with EtOAc and washed with 10% citric acid (2×), saturatedNaHCO₃ (2×) and brine. The organic layer was collected, dried andconcentrated under reduced pressure to provide 6-1c.

Step b)

The product from step a) was treated with 10 ml of 50% CH₂Cl₂/TFA for 1hr at room temperature to yield 6-1d as its TFA salt. MS (m/z) M+1=493.Step Three

Step a)

Intermediate 6-1d (200 mg, 0.33 mmol), DMAP (5 mg, catalytic) and DIPEA(230 μL, 1.32 mmol) were dissolved in dry dichloromethane (5 mL) underN₂ and terephtaloyl chloride (30 mg, 0.15 mmol) was then added and thesolution was stirred for 24 h at RT. The contents were then added to aseparatory funnel along with EtOAc and washed with 10% citric acid (2×),saturated NaHCO₃ (2×) and brine. The organic layer was collected, driedand concentrated under reduced pressure to yield the product 6-1e as ayellow oil.

Step b)

6-1e (160 mg, 0.19 mmol) and 10% Pd/C (50% H₂O, 100 mg) were mixedtogether in MeOH (10 ml) under N₂, the N₂ is then flushed with H₂ andthe mixture was stirred for 24 h at RT. The mixture is filtered oncelite, washed with MeOH. The filtrate was collected, dried andconcentrated under reduced pressure to yield the product 6-1f. MS (m/z)M+1=847.Step Four

Step a)

Boc-N-Me-Ala-OH (74 mg, 0.37 mmol), HOBt (59 mg, 0.38 mmol), HBTU (144mg, 0.38 mmol) and DIPEA (140 μl, 0.8 mmol) were dissolved in dry DMF (5ml) under N₂ and stirred for 10 min at RT. 6-1f (135 mg, 0.16 mmol) wasthen added and the solution was left to stir for 24 h at RT. Thecontents were then added to a separatory funnel along with ETOAc andwashed with 10% citric acid (2×), saturated NaHCO₃ (2×) and brine. Theorganic layer was collected, dried and concentrated under reducedpressure to provide 6-1g.

Step b)

Intermediate 6-1g was subsequently treated with 4N HCl in 1,4-dioxanefor 1 hr at room temperature. Trituration with diethyl ether providedthe bis-HCl salt of compound 15. MS (m/z) M+1=1017.7. Synthesis of Compound 14

Step a)

To a solution of 7-1a (206 mg, 0.35 mmol) in dichloromethane (5 mL) weresequentially added DIPEA (100 uL, 0.57 mmol) and terephthaloyl chloride(31.3 mg, 0.15 mmol) and the reaction was stirred for 12 hrs at roomtemperature. Water and ethyl acetate were added, the organic layer wasseparated, washed with 10% citric acid, aqueous NaHCO₃ and brine, driedover MgSO₄ and concentrated under reduced pressure. Purification byflash chromatography provided 7-1b as a white solid.

Step b)

A 4 N solution of HCl in 1,4-dioxane (1 mL) was added to 7-1b (16 mg,0.01 mmol) at room temperature and the solution was stirred for 2 h andthen concentrated in vacuum. Trituration with diethyl ether providedcompound 14•2HCl as a white solid, MS (m/z) (M+2)/2=546.5.

8. Synthesis of Compound 23

Step a)

Intermediate 1-2d (250 mg, 0.78mmol), HOBt (120 mg, 0.78 mmol), HBTU(300 mg, 0.78 mmol) and DIPEA (525 μL, 3 mmol) were dissolved in dry DMF(5 mL) under N₂ and stirred for 10 min at room temperature. Intermediate6-1b (215 mg, 0.6 mmol) was added and the solution was left to stir for24 h at room temperature. The contents were added to a separatory funnelalong with EtOAc and washed with 10% citric acid (2×), brine (2×) andsaturated NaHCO₃ (2×). The organic layer was collected, dried andconcentrated under reduced pressure. The product was purified by flashchromatography (hexanes/EtOAc) and subsequently treated with 4N HCl in1,4-dioxane, volatiles were removed and trituration with diethyl etherprovides 8-1a as the HCl salt. MS (m/z) M+1=384.3.Step b)

Boc-Me-Ala-OH (130 mg, 0.63 mmol), HOBt (100 mg, 0.63 mmol), HBTU (240mg, 0.63 mmol) and DIPEA (420 μL, 2.4 mmol) were dissolved in dry DMF (5mL) under N₂ and stirred for 10 min at RT. 8-1b (200 mg, 0.48 mmol) wasthen added and the solution was left to stir for 24 h at RT. Thecontents were then added to a separatory funnel along with EtOAc andwashed with 10% citric acid (2×), brine (2×) and saturated NaHCO₃ (2×).The organic layer was collected, dried and concentrated under reducedpressure. The product 8-1b was purified by flash chromatography(hexanes/EtOAc). MS (m/z) M+1=569.4Step c)

Intermediate 8-1b (70 mg, 0.123 mmol), CuCl (20 mg, 0.185 mmol) andtetramethylethylenediamine (27 μL, 0.185 mmol) were dissolved in dryacetone (3 mL) and stirred at RT under an O₂ atmosphere for 72 h. EtOAcwas added and the mixture was transferred to a separatory funnel. Themixture was washed with 10% citric acid (2×), brine (2×) and saturatedNaHCO₃ (2×). The organic layer was collected, dried and concentratedunder reduced pressure. The product was purified by flash chromatography(hexanes/THF). The resulting product was stirred with 4N HCl in1,4-dioxane for 2 hrs. Volatiles were removed under reduced pressure andthe residue triturated with diethyl ether to provide compound 23 as itsbis-HCl salt. MS (m/z) M+1=935.1.9. Synthesis of Compound 25

To a solution of 23.2HCl (100 mg, 0.1 mmol) in anhydrous MeOH (10 mL)and stirred under N₂ was added 10% Pd/C (500 mg). The reaction mixturewas purged with hydrogen and stirred for 16 hr under atmosphericpressure of hydrogen. The mixture was then filtered through celite andthe filtrate was concentrated in vacuo to provide compound 25•2HCl as awhite solid. MS (m/z) M+1=943.6.10. Synthesis of Compound 41

Step a)

To a solution of 10-a (4.90 g, 6.15 mmol) in anhydrous MeOH (120 mL) andstirred under N₂ was added 10% Pd/C (500 mg). The reaction mixture waspurged with H₂ and stirred for 3 hr, then filtered through celite. Thefiltrate was concentrated in vacuo to provide intermediate 10-b as awhite solid. MS (m/z) M+1=662.4.

Step b)

A solution of 10-b (200 mg, 0.30 mmol) in dichloromethane, cooled to 0°C., were sequentially added Et₃N (84 μl, 0.60 mmol) and oxalyl chloride(13 μl, 0.15 mmol). The reaction was then stirred for 4 hrs at roomtemperature. Aqueous NaHCO₃ and ethyl acetate were added, the organiclayer was separated, washed with brine, dried over anhydrous MgSO₄,filtered and concentrated in vacuo. Purification by silica gelchromatography eluting with a hexane/tetrahydrofuran gradient providedthe expected compound 10-c as a white solid.

Step c)

4N HCl in 1,4-dioxane (3 ml) was added to 10-c (95 mg, 0.07 mmol) andthe solution was stirred for 2 hrs at room temperature. Volatiles wereremoved under reduced pressure and the residue was triturated withdiethyl ether to provide compound 41 as its bis-HCl salt. MS (m/z)(M+2)/2=589.4.

Representative compounds of the present invention were prepared bysimple modification of the above procedures and are illustrated in Table1: TABLE 1 Compound Structure (M + 2)/2 1

524.6 2

605.5 3

532.6 4

568.4 5

470.4 6

582.5 7

562.4 8

506.4 9

562.4 10

575.4 11

526.3 12

562.4 13

543.5 14

546.5 15

509.6 16

457.4 17

565.4 18

527.3 19

534.5 20

541.4 21

548.3 22

555.5 23

935.1 (M + 1) 24

510.2 25

942.6 (M + 1) 26

498.6 27

499.6 28

506.4 29

488.4 30

562.7 31

569.6 32

513.4 33

527.4 34

541.4 35

555.4 36

528.4 37

510.4 38

537.4 39

524.4 40

603.4 41

589.4 42

575.4 43

610.4 44

617.4 45

627.4 46

541.4 47

527.4

Representative compounds of the present invention which can be preparedby simple modification of the above procedures are illustrated in Tables2 through 11: TABLE 2 M1-BG-M2 Formula 1A BG is

M1 M2

A = CH₂; R³ = H or Me A = CH₂; R³ = H or Me

A = CH₂; R³ = H or Me A = CH₂; R³ = H or Me

A = CH₂; R³ = H or Me A = CH₂; R³ = H or Me

A = CH₂; R³ = H or Me A = CH₂; R³ = H or Me

A = CH₂; R³ = H or Me A = CH₂; R³ = H or Me

A = CH₂; R³ = H or Me A = CH₂; R³ = H or Me

A = CH₂; R³ = H or Me A = CH₂; R³ = H or Me

A = CH₂; R³ = H or Me A = CH₂; R³ = H or Me

A = CH₂; R³ = H or Me A = CH₂; R³ = H or Me

A = CH₂; R³ = H or Me A = CH₂; R³ = H or Me

A = CH₂; R³ = H or Me A = CH₂; R³ = H or Me

A = CH₂; R³ = H or Me A = CH₂; R³ = H or Me

A = CH₂; R³ = H or Me A = CH₂; R³ = H or Me

A = CH₂; R³ = H or Me A = CH₂; R³ = H or Me

A = CH₂; R³ = H or Me A = CH₂; R³ = H or Me

A = CH₂; R³ = H or Me A = CH₂; R³ = H or Me

A = CH₂; R³ = H or Me A = CH₂; R³ = H or Me

A = CH₂; R³ = H or Me A = CH₂; R³ = H or Me

A = CH₂; R³ = H or Me A = CH₂; R³ = H or Me

A = CH₂; R³ = H or Me A = CH₂; R³ = H or Me

A = CH₂; R³ = H or Me A = CH₂; R³ = H or Me

A = CH₂; R³ = H or Me A = CH₂; R³ = H or Me

A = CH₂; R³ = H or Me A = CH₂; R³ = H or Me

A = CH₂; R³ = H or Me A = CH₂; R³ = H or Me

A = CH₂; R³ = H or Me A = CH₂; R³ = H or Me

A = CH₂; R³ = H or Me A = CH₂; R³ = H or Me

A = CH₂; R³ = H or Me A = CH₂; R³ = H or Me

A = CH₂; R³ = H or Me A = CH₂; R³ = H or Me

A = CH₂; R³ = H or Me A = CH₂; R³ = H or Me

A = CH₂; R³ = H or Me A = CH₂; R³ = H or Me

A = CH₂; R³ = H or Me A = CH₂; R³ = H or Me

A = CH₂; R³ = H or Me A = CH₂; R³ = H or Me

A = CH₂; R³ = H or Me A = CH₂; R³ = H or Me

A = CH₂; R³ = H or Me A = CH₂; R³ = H or Me

A = CH₂; R³ = H or Me A = CH₂; R³ = H or Me

A = CH₂; R³ = H or Me A = CH₂; R³ = H or Me

A = CH₂; R³ = H or Me A = CH₂; R³ = H or Me

A = CH₂; R³ = H or Me A = CH₂; R³ = H or Me

A = CH₂; R³ = H or Me A = CH₂; R³ = H or Me

A = CH₂; R³ = H or Me A = CH₂; R³ = H or Me

A = CH₂; R³ = H or Me A = CH₂; R³ = H or Me

A = CH₂; R³ = H or Me A = CH₂; R³ = H or Me

A = CH₂; R³ = H or Me A = CH₂; R³ = H or Me

A = CH₂; R³ = H or Me A = CH₂; R³ = H or Me

A = CH₂; R³ = H or Me A = CH₂; R³ = H or Me

A = CH₂; R³ = H or Me A = CH₂; R³ = H or Me

A = CH₂; R³ = H or Me A = CH₂; R³ = H or Me

A = CH₂; R³ = H or Me A = CH₂; R³ = H or Me

A = CH₂; R³ = H or Me A = CH₂; R³ = H or Me

A = CH₂; R³ = H or Me A = CH₂; R³ = H or Me

A = CH₂; R³ = H or Me A = CH₂; R³ = H or Me

A = CH₂; R³ = H or Me A = CH₂; R³ = H or Me

A = CH₂; R³ = H or Me A = CH₂; R³ = H or Me

A = CH₂; R³ = H or Me A = CH₂; R³ = H or Me

A = CH₂; R³ = H or Me A = CH₂; R³ = H or Me

A = CH₂; R³ = H or Me A = CH₂; R³ = H or Me

A = CH₂; R³ = H or Me A = CH₂; R³ = H or Me

A = CH₂; R³ = H or Me A = CH₂; R³ = H or Me

A = CH₂; R³ = H or Me A = CH₂; R³ = H or Me

A = CH₂; R³ = H or Me A = CH₂; R³ = H or Me

A = CH₂; R³ = H or Me A = CH₂; R³ = H or Me

A = CH₂; R³ = H or Me A = CH₂; R³ = H or Me

A = CH₂; R³ = H or Me A = CH₂; R³ = H or Me

A = CH₂; R³ = H or Me A = CH₂; R³ = H or Me

A = CH₂; R³ = H or Me A = CH₂; R³ = H or Me

A = CH₂; R³ = H or Me A = CH₂; R³ = H or Me

A = CH₂; R³ = H or Me A = CH₂; R³ = H or Me

A = CH₂; R³ = H or Me A = CH₂; R³ = H or Me

A = CH₂; R³ = H or Me A = CH₂; R³ = H or Me

A = CH₂; R³ = H or Me A = CH₂; R³ = H or Me

A = CH₂; R³ = H or Me A = CH₂; R³ = H or Me

A = CH₂; R³ = H or Me A = CH₂; R³ = H or Me

A = CH₂; R³ = H or Me A = CH₂; R³ = H or Me

A = CH₂; R³ = H or Me A = CH₂; R³ = H or Me

A = CH₂; R³ = H or Me A = CH₂; R³ = H or Me

A = CH₂; R³ = H or Me A = CH₂; R³ = H or Me

A = CH₂; R³ = H or Me A = CH₂; R³ = H or Me

A = CH₂; R³ = H or Me A = CH₂; R³ = H or Me

A = CH₂; R³ = H or Me A = CH₂; R³ = H or Me

A = CH₂; R³ = H or Me A = CH₂; R³ = H or Me

A = CH₂; R³ = H or Me A = CH₂; R³ = H or Me

A = CH₂; R³ = H or Me A = CH₂; R³ = H or Me

A = CH₂; R³ = H or Me A = CH₂; R³ = H or Me

A = CH₂; R³ = H or Me A = CH₂; R³ = H or Me

A = CH₂; R³ = H or Me A = CH₂; R³ = H or Me

A = CH₂; R³ = H or Me A = CH₂; R³ = H or Me

A = CH₂; R³ = H or Me A = CH₂; R³ = H or Me

A = CH₂; R³ = H or Me A = CH₂; R³ = H or Me

A = CH₂; R³ = H or Me A = CH₂; R³ = H or Me

A = CH₂; R³ = H or Me A = CH₂; R³ = H or Me

A = CH₂; R³ = H or Me A = CH₂; R³ = H or Me

A = CH₂; R³ = H or Me A = CH₂; R³ = H or Me

A = CH₂; R³ = H or Me A = CH₂; R³ = H or Me

A = CH₂; R³ = H or Me A = CH₂; R³ = H or Me

A = CH₂; R³ = H or Me A = CH₂; R³ = H or Me

A = CH₂; R³ = H or Me A = CH₂; R³ = H or Me

A = CH₂; R³ = H or Me A = CH₂; R³ = H or Me

A = CH₂; R³ = H or Me A = CH₂; R³ = H or Me

A = CH₂; R³ = H or Me A = CH₂; R³ = H or Me

A = CH₂; R³ = H or Me A = CH₂; R³ = H or Me

A = CH₂; R³ = H or Me A = CH₂; R³ = H or Me

A = CH₂; R³ = H or Me A = CH₂; R³ = H or Me

A = CH₂; R³ = H or Me A = CH₂; R³ = H or Me

A = CH₂; R³ = H or Me A = CH₂; R³ = H or Me

A = CH₂; R³ = H or Me A = CH₂; R³ = H or Me

A = CH₂; R³ = H or Me A = CH₂; R³ = H or Me

A = CH₂; R³ = H or Me A = CH₂; R³ = H or Me

A = CH₂; R³ = H or Me A = CH₂; R³ = H or Me

A = CH₂; R³ = H or Me A = CH₂; R³ = H or Me

A = CH₂; R³ = H or Me A = CH₂; R³ = H or Me

A = CH₂; R³ = H or Me A = CH₂; R³ = H or Me

A = CH₂; R³ = H or Me A = CH₂; R³ = H or Me

A = CH₂; R³ = H or Me A = CH₂; R³ = H or Me

A = CH₂; R³ = H or Me A = CH₂; R³ = H or Me

A = CH₂; R³ = H or Me A = CH₂; R³ = H or Me

A = CH₂; R³ = H or Me A = CH₂; R³ = H or Me

A = CH₂; R³ = H or Me A = CH₂; R³ = H or Me

A = CH₂; R³ = H or Me A = CH₂; R³ = H or Me

A = CH₂; R³ = H or Me A = CH₂; R³ = H or Me

A = CH₂; R³ = H or Me A = CH₂; R³ = H or Me

A = CH₂; R³ = H or Me A = CH₂; R³ = H or Me

TABLE 3 M1-B-BG-B¹-M2 Formula 1B BG is

B and B are C₁-C₆ alkyl M1 M2

A = CH₂ A = CH₂

A = CH₂ or C = O A = CH₂ or C = O

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ or C = O A = CH₂ or C = O

A = CH₂ A = CH₂

A = CH₂ or C = O A = CH₂ or C = O

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ or C = O A = CH₂ or C = O

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ or C = O A = CH₂ or C = O

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ or C = O A = CH₂ or C = O

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ or C = O A = CH₂ or C = O

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ or C = O A = CH₂ or C = O

A = CH₂ or C = O A = CH₂ or C = O

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ or C = O A = CH₂ or C = O

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂Note: In M1 and M2, the stereochemistry at the connecting carbon is (S)

TABLE 4 M1-B-BG-B¹-M2 Formula 1B BG is

B and B¹ are C₁-C₆ alkyl M1 M2

A = CH₂ A = CH₂

A = CH₂ or C = O A = CH₂ or C = O

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ or C = O A = CH₂ or C = O

A = CH₂ A = CH₂

A = CH₂ or C = O A = CH₂ or C = O

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ or C = O A = CH₂ or C = O

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ or C = O A = CH₂ or C = O

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ or C = O A = CH₂ or C = O

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ or C = O A = CH₂ or C = O

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ or C = O A = CH₂ or C = O

A = CH₂ or C = O A = CH₂ or C = O

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ or C = O A = CH₂ or C = O

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂

A = CH₂ A = CH₂Note: In M1 and M2, the stereochemistry at the connecting carbon is (S)

TABLE 5 M1—BG—M2 Formula 1A

M1 M2

TABLE 6 M1—B—BG—B¹—M2 Formula 1B

M1 M2

TABLE 7 M1—B—BG—B¹—M2 Formula 1B

M1 M2

TABLE 8

R⁴ R⁴⁰⁰

TABLE 9

R⁴

TABLE 10

R¹¹

TABLE 11

R¹¹

Assays11. Molecular Constructs for Expression

GST-XIAP BIR3RING: XIAP coding sequence amino acids 246-497 cloned intoPGEX2T1 via BamHI and AVA I. The plasmid was transformed into E. coliDH5α for use in protein expression and purification.

GST-HIAP2 (cIAP-1) BIR 3: HIAP2 coding sequence from amino acids 251-363cloned into PGex4T3 via BamHI and XhoI. The plasmid was transformed intoE. coli DH5α for use in protein expression and purification.

GST-HIAP1(cIAP-2) BIR 3: HIAP1 coding sequence from amino acids 236-349,cloned into PGex4T3 via BamHI and XhoI. The plasmid was transformed intoE. coli DH5α for use in protein expression and purification.

GST-linker BIR 2 BIR3Ring: XIAP coding sequence from amino acids 93497cloned into PGex4T1 via BamHI and XhoI. Amino acids 93-497 wereamplified from full length XIAP in pGex4t3, using the primers:TTAATAGGATCCATCAACGGCTTTTATC and GCTGCATGTGTGTCAGAGG, using standard PCRconditions. The PCR fragment was TA cloned into pCR-2.1 (invitrogen).Linker BIR 2 BIR 3Ring was subcloned into pGex4T1 by BamHI/XhoIdigestion. The plasmid was transformed into E. coli DH5α for use inprotein expression and purification.

Full-length human XIAP, Aegera plasmid number 23. XIAP coding sequenceamino acids 1-497 cloned into GST fusion vector, PGEX4T1 via BamHI andXhoI restriction sites. (a gift from Bob Korneluk and Peter Liston). Theplasmid was transformed into E. coli DH5α for use in proteinpurification.

GST-XIAP linker BIR 2: XIAP linker BIR 2 coding sequence from aminoacids 93-497 cloned into pGex4T3 via BamHI and XhoI. The plasmid wastransformed into E. coli DH5α for use in protein expression andpurification.

12. Synthesis of Fluorescent Probe for FP Assay

A fluorescent peptide probe,Fmoc-Ala-Val-Pro-Phe-Tyr(t-Bu)-Leu-Pro-Gly(t-Bu)-Gly-OH was preparedusing standard Fmoc chemistry on 2-chlorotrityl chloride resin (Int. J.Pept. Prot. Res. 38:555-561, 1991). Cleavage from the resin wasperformed using 20% acetic acid in dichloromethane (DCM), which left theside chain still blocked. The C-terminal protected carboxylic acid wascoupled to 4′-(aminomethy)fluorescein (Molecular Probes, A-1351; Eugene,Oreg.) using excess diisopropylcarbodiimide (DIC) in dimethylformamide(DMF) at room temperature and was purified by silica gel chromatography(10% methanol in DCM). The N-terminal Fmoc protecting group was removedusing piperidine (20%) in DMF, and purified by silica gel chromatography(20% methanol in DCM, 0.5% HOAc). Finally, the t-butyl side chainprotective groups were removed using 95% trifluoroacetic acid containing2.5% water and 2.5% triisopropyl silane. The peptide obtained displayeda single peak by HPLC (>95% pure).

13. Expression and Purification of Recombinant Proteins

A. Recombinant Proteins Expression

Glutathione S-transferase (GST) tagged proteins were expressed inEscherichia coli strains DH5-alpha. For expression full length XIAP,individual or combinations of XIAP-BIR domains, cIAP-1, cIAP-2 and Livintransformed bacteria were cultured overnight at 37° C. in Luria Broth(LB) medium supplemented with 50 ug/ml of ampicillin. The overnightculture was then diluted 25 fold into fresh LB ampicillin supplementedmedia and bacteria were grown up to A₆₀₀=0.6 then induced with 1 mMisopropyl-D-1-thiogalactopyranoside for 3 hours. Upon induction, cellswere centrifuged at 5000 RPM for 10 minutes and the media was removed.Each pellet obtained from a 1 liter culture received 10 ml of lysisbuffer (50 mM Tris-HCl, 200 mM NaCl, 1 mM DTT, 1 mM PMSF, 2 mg/ml oflysosyme, 100 μg/ml)), was incubated at 4° C. with gentle shaking. After20 minutes of incubation, the cell suspension was placed at −80° C.overnight or until needed.

B. Purification of Recombinant Proteins

For purification of recombinant proteins, the IPTG-induced cell lysatewas thawed vortexed and then disrupted by flash freezing in liquidnitrogen two times with vortexing after each thaw. The cells weredisrupted further by passing the extract four times through a Bio-NebCell disruptor device (Glas-col) set at 100 psi with Nitrogen gas. Theextract was clarified by centrifugation at 4 C at 15000 RPM in a SS-34Beckman rotor for 30 minutes. The resulting supernatant was then mixedwith 2 ml of glutathione-Sepharose beads (Pharmacia) per 500 ml cellculture (per 1000ml culture for full length XIAP) for 1 hour at 4 C.Afterwards, the beads were washed 3 times with 1× Tris-Buffered Saline(TBS) to remove unbound proteins. The retained proteins were eluted with2 washes of 2 ml of 50 mM TRIS pH 8.0 containing 10 mM reducedglutathione. The eluted proteins were pooled and precipitated with 604g/liter of ammonium sulfate and the resulting pellet re-suspended intoan appropriate buffer. As judged by SDS-PAGE the purified proteinswere >90% pure. The protein concentration of purified proteins wasdetermined from the Bradford method.

His-tag proteins were expressed in the E. Coli strain in E. coli AD494cells using a pet28ACPP32 construct. The soluble protein fraction wasprepared as described above. For protein purification, the supernatantwas purified by affinity chromatography using chelating-Sepharose(Pharmacia) charged with NiSO₄ according to the manufacturer'sinstructions.

Purity of the eluted protein was >90% pure as determined by SDS-PAGE.The protein concentration of purified proteins was determined from theBradford assay.

Binding Assay

14. Fluorescence Polarization-Based Competition Assay

For all assays, the fluorescence and fluorescence-polarization wasevaluated using a Tecan Polarion instrument with the excitation filterset at 485 nm and the emission filter set at 535 nm. For each assay, theconcentration of the target protein was first establish by titration ofthe selected protein in order to produce a linear dose-response signalwhen incubated alone in the presence of the fluorescent probe. Uponestablishing these conditions, the compounds potency (IC₅₀) andselectivity, was assessed in the presence of a fix defined-amount oftarget protein and fluorescent probe and a 10 point serial dilution ofthe selected compounds. For each IC₅₀ curve, the assays were run asfollowed: 25 ul/well of diluted compound in 50 mM MES buffer pH 6.5 wereadded into a black 96 well plate then 25 ul/well of bovine serum albumin(BSA) at 0.5 mg/ml in 50 mM MES pH 6.5. Auto-fluorescence for eachcompound was first assessed by performing a reading of the compound/BSAsolution alone. Then 25 ul of the fluorescein probe diluted into 50 mMMES containing 0.05 mg/ml BSA were added and a reading to detectquenching of fluorescein signal done. Finally 25 ul/well of the targetor control protein (GST-BIRs) diluted at the appropriate concentrationin 50 mM MES containing 0.05 mg/ml BSA were added and the fluorescencepolarization evaluated.

15. Determination of IC₅₀ and Inhibitory Constants

For each assay the relative polarization-fluorescence units were plottedagainst the final concentrations of compound and the IC₅₀ calculatedusing the Grad pad prism software and/or Cambridge soft. The ki valuewere derived from the calculated IC₅₀ value as described above andaccording to the equation described in Nikolovska-Coleska, Z. (2004)Anal Biochem 332, 261-273.

16. Caspase-3 Full Length XIAP, Linker BIR2 or Linker-BIR2-BIR3-RINGDerepression Assay

In order to determine the relative activity of the selected compoundagainst XIAP-Bir2, we setup an in vitro assay where caspase-3 wasinhibited by GST fusion proteins of XIAP linker-bir2, XIAP LinkerBir2-Bir3-RING or full-length XIAP. Caspase 3 (0.125 ul) and 12.25-34.25nM (final concentration) of GST-XIAP fusion protein (GST-Bir2,GST-Bir2Bir3RING or full-length XIAP) were co-incubated with serialdilutions of compound (200 uM-5 pM). Caspase 3 activity was measured byoverlaying 25 ul of a 0.4 mM DEVD-AMC solution. Final reaction volumewas 100 ul. All dilutions were performed in caspase buffer (50 mM HepespH 7.4, 100 mM NaCl, 10% sucrose, 1 mM EDTA, 10 mM DTT, 0.1% CHAPS(Stennicke, H. R., and Salvesen, G. S. (1997). Biochemicalcharacteristics of caspase-3, -6, -7, and -8. J. Biol. Chem. 272,25719-25723)

The fluorescent AMC released from the caspase-3 hydrolysis of thesubstrate was measured in a TECAN spectrophotometer at 360 nm excitationand 444 nm emission, after 15 minutes of incubation at room temperature.IC50 values were calculated on a one or two-site competition model usingGraphPad v4.0, using the fluorescence values after 15 minutes ofincubation plotted against the log10 concentration of compound.

The compounds which were tested in the apoptosome assay and thelinker-BIR2-Bir3/caspase-3 inhibition assay were found to have IC₅₀s asillustrated in Table 12. TABLE 12 In vitro activitity of selectedcompounds against IAP's. Apoptosome Fluorescent polarization L-Bir2-Bir3(FP) assay Cpd# XIAP Xiap cIAP-1 cIAP-2 Number μM nM nM nM 1 A B C C 2 AB B C 3 B B B A 4 B A nd nd 6 B A nd nd 7 B B nd nd 8 B A nd nd 11 C ndnd C 12 C C B A 13 nd B nd nd 14 nd B nd nd 15 nd nd nd nd 23 B B A Bnd = Not determined;Legend:FP assay: A ≦ 5 nM; B ≦ 100 nM; C ≧ 100 nM;Legend: Apoptosome assay: A ≦ 0.1 μM; B ≦ 0.5 μM; C ≧ 1 μM

Results demonstrate that the selected compounds can inhibit thecaspase-blocking activity of XIAP in an apoptosome assay (express ineffective concentration to achieve 50% of activation and report the Kito bind to various IAP's. This Ki was calculated from the displacement afluorescent probe capable to bind to the bir3 domain of various IAP'susing a fluorescent polarization assay.

Cell-Free Assay

17. Caspase De-Repression Assay using Cellular Extracts (Apoptosome)

100 ug of 293 cell S100 extract and 0.25 uM-2 uM of GST-XIAP fusionprotein (XIAP-Bir3RING, XIAP-Linker Bir2Bir3RING, or full-length XIAP)were co-incubated with serial dilutions of compound (40 uM-5 pM).Caspases present in the extracts were activated by adding 1 mM dATP, 0.1mM ALLN, 133 ug Cytochrome C (final concentrations), and incubating at37° C. for 25 minutes. All reactions and dilutions used S100 buffer (50mM Pipes pH 7.0, 50 mM KCl, 0.5 mM EGTA pH 8.0, 2 mM MgCl₂ supplementedwith 1/1000 dilutions of 2 mg/ml Cytochalisin B, 2 mg/ml Chymotstatin,Leupeptin, Pepstatin, Antipain, 0.1M PMSF, 1M DTT). Final reactionvolume was 30 ul. Caspase-3 activity was measured by overlaying 30 ul ofa 0.4 mM DEVD-AMC solution. released AMC cleavage was measured in aTECAN spectrophotometer at 360 nm excitation and 444 nm emission, on akinetic cycle of 1 hour with readings taken every 5 minutes. Caspaseactivity was calculated as V_(o) of AMC fluorescence/sec. Caspasede-repression by our compounds was compared to fully activated extractand activated extract repressed by the presence of XIAP fusion protein.

18. Cell Culture and Cell Death Assays

A. Cell Culture

MDA-MD-231 (breast) and SKOV-3 (ovarian) cancer cells were cultured inRPMI1640 media supplemented with 10% FBS and 100 units/mL of Penicillinand Streptomycin.

B. Assays

Viability assays were done on a number of cells including MDA-MB-231,SKOV-3, H460, PC3, HCT-116, and SW480 cells. Cells were seeded in 96well plates at a respective density of 5000 and 2000 cells per well andincubated at 37° C. in presence of 5% CO₂ for 24 hours. Selectedcompounds were diluted into the media at various concentration rangingfrom 0.01 uM up to 100 uM. Diluted compounds were added onto theMDA-MB-231 cells. For the MDA-MB-231 SKOV3, H460, PC3, HCT-116, andSW480 cells, the compounds were added either alone or in presence of 1-3ng/ml of TRAIL. After 72 hours cellular viability was evaluated by MTTbased assays. IC₅₀s of select compounds against MDA and SKOV3 cell linesare presented in Table 13: TABLE 13 IC₅₀s of select compounds againstMDA and SKOV3 cell lines MDA SKOV3 Compound EC₅₀ (nM) EC₅₀ (nM) 1 A A 2A 3 A 4 A 5 A 6 A 7 A 8 A 9 A 10 A 11 A 12 A 13 B 14 B 15 A 16 A 17 B 18B 19 B 20 B 21 B 22 A 23 A A 24 A 25 A 26 A 27 B 28 A 29 A 30 A 31 A 32C 33 C 34 C 35 B B 36 A 37 A 38 A 39 B 40 B 41 B 42 C 43 B 44 B 45 B 46A 47 B

The compounds exemplified in Table 1 were tested and found to have IC₅₀sin the following ranges: A>100 nM; B>1000 nM; C>1000 nM.

19. Apoptosis Assay: Measurement of Caspase-3 Activity from CulturedCells.

One day, prior to the treatment, 10 000 cells per well were plated in awhite tissue culture treated 96 well plate with 100 ul of media. On theday of compound treatment, compounds were diluted with cell culturemedia to a working stock concentration of 2× and 100 ul of dilutedcompound were added to each well and the plate was incubated for 5 h at37° C. in presence of 5% CO₂. Upon incubation, the plate was washedtwice with 200 ul of cold TRIS Buffered Saline (TBS) buffer. Cells werelysed with 50 ul of Caspase assay buffer (20 mM Tris-HCl pH 7.4, 0.1%NP-40, 0.1% Chaps, 1 mM DTT, 0.1 mM EDTA, 0.1 mM PMSF, 2 mg/mlChymotstatin, Leupeptin, Pepstatin, Antipain) then incubated at 4° C.with shaking for 30 minutes. 45 ul of Caspase assay buffer and 5 ul ofAc-DEVD-AMC at 1 mg/ml were added to each well, the plate shaken andincubated for 16 h at 37° C. The amount of release AMC was measured in aTECAN spectrophotometer at with the excitation and emission filter setat 360 nm and 444 nm. The percentage of Caspase-3 activity was expressedin comparison of the signal obtained with the non-treated cells.

20. Cellular Biochemistry:

A. Detection of XIAP and PARP/Caspase-3/Caspase-9

Detection of cell expressed XIAP and PARP were done by western blotting.Cells were plated at 300 000 cells/well in a 60 mm wells (6 wells platedish). The next day the cells were treated with selected compound at theindicated concentration. 24 hours later cells the trypsinized cells,pelleted by centrifugation at 1800 rpm at 4° C. The resulting pellet wasrinsed twice with cold TBS. The final washed pellet of cells was thelysed with 250 ul Lysis buffer (NP-40, glycerol, 1% of a proteaseinhibitor cocktail (Sigma)), placed at 4° C. for 25 min with gentleshaking. The cells extract was centrifuged at 4° C. for 10 min at 10 000rpm. Both the supernatant and the pellet were kept for western blottinganalysis as described below. From the supernatant, the protein contentwas evaluated and about 50 ug of protein was fractionated onto a 10%SDS-PAGE. Pellets were washed with the lysis buffer and re-suspend into50 ul of Lamelli buffer 1×, boiled and fractionated on SDS-PAGE. Uponelectrophoresis each gel was electro-transferred onto a nitrocellulosemembrane at 0.6 A for 2 hours. Membrane non-specific sites were blockedfor 1 hours with 5% Skim milk in TBST (TBS containing 0.1% (v/v)Tween-20) at RT. For protein immuno-detection, membranes were incubatedovernight with primary antibodies raised against XIAP clone 48 obtainedfrom Becton-Dickison) or PARP: obtained from Cell signal or caspase-3 orcaspase-9 primary antibodies were incubated at 4° C. with shaking atdilutions as follows: XIAP clone 80 (Becton-Dickinson) 1/2500 PARP (CellSignal) 1/2500 Caspase 3 (Sigma) 1/1500 Caspase 9 (Upstate) 1/1000

Upon overnight incubation, the membranes received three washes of 15 minin TBST then were incubated for 1 hour at room temperature in thepresence of a secondary antibody coupled with HRP-enzyme (Chemicon) anddiluted at 1/5000. Upon incubation each membrane were washed three timeswith TBST and the immunoreactive bands were detected by addition of aluminescent substrate (ECL kit Amersham) and capture of signal on aX-RAY film for various time of exposure. Active compounds were shown toinduce the cleavage of PARP and XIAP as well as to translocate XIAP intoan insoluble compartment.

21. Hollow Fiber Model

Hollow fiber in vivo model were used to demonstrate in vivo efficacy ofselected compounds against selected cell lines as single agent therapyor in combination with selected cytotoxic agents. At day 1, selectedcell lines were cultured and the fiber filled at a cell density of about40,000 cells/fiber. At the day of operation (day 4), three fibers areimplanted sub-cutaneous into 28-35 g Nu/Nu CD-1 male mice. On day 5,mice start to receive daily injection via Intravenous or sub-cutaneousroute of control vehicle or vehicle containing the selected compound atthe appropriate concentration and/or injection of cytotoxic agent viaintra-peritoneal route. Upon 7 days of non-consecutive treatments, theanimals are sacrificed, each fiber is removed and the metabolicviability of the remaining cells determined by MTT assay. Efficacy ofthe compound is define as the difference between the MTT values obtainedfrom the cell containing fiber taken from the vehicle-treated animal andthe from the animal treated with the compound alone or the compoundgiven in combination of the cytotoxic agent

22. Combination Anti-Cancer Therapy in Vivo

Female nude mice received 2×10 HCT-116 subdermally on the right flank.On day 26, when tumors were ˜90 mm, animals were assigned to groupsusing a balanced design based on tumor size. At that time mitomycin-Cand Compound 23 treatment was initiated. Mitomycin-C was administered ipat 1 mg/kg, Monday through Friday for two weeks. Compound 23 was giveniv at 1 or 5mg/kg five times per week for the duration of theexperiment. Tumor measurements were taken twice weekly. As illustratedin FIG. 1, compound 23 showed an increasing anti-tumor effect incombination with mitomycin-C with increasing dose, with 5 mg/kg showingsuperior anti-tumor effects compared to the 1 mg/kg dose.

23. Pharmacokinetic Studies

Selected compounds were dissolved into normal saline or appropriatevehicle and given at various doses using different route ofadministration, including intravenous bolus, intravenous infusion, oraland subcutaneous injection.

All literature, patents, published patent applications cited herein arehereby incorporated by reference.

From the foregoing, it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention. Accordingly, the invention is notlimited except as by the appended claims.

1. A compound represented by formula:

or a salt thereof, wherein: m is 0, 1 or 2; p is 1 or 2; Y is NH, O orS; A and A¹ are independently selected from 1) —CH₂—, 2) —CH₂CH₂—, 3)—C(CH₃)₂—, 4) —CH(C₁-C₆ alkyl)-, 5) —CH(C₃-C₇ cycloalkyl)-, 6) —C₃-C₇cycloalkyl-, 7) —CH(C₁-C₆ alkyl-C₃-C₇ cycloalkyl)-, or 8) —C(O)—; B andB¹ are independently C₁-C₆ alkyl; X and X¹ are independently selectedfrom

R¹, R¹⁰⁰, R² and R²⁰⁰ are independently selected from: 1) H, or 2) C₁-C₆alkyl optionally substituted with one or more R⁶ substituents; Q and Q¹are each independently 1) NR⁴R⁵, 2) OR¹¹, or 3) S(O)_(m)R¹¹; or Q and Q¹are each independently

wherein G is a 5, 6 or 7 membered ring which optionally incorporates oneor more heteroatoms chosen from S, N or O, the ring being optionallysubstituted with one or more R¹² substituents; R⁴ and R⁵ are eachindependently 1) H, 2) haloalkyl, 3) ←C₁-C₆ alkyl, 4) ←C₂-C₆ alkenyl, 5)←C₂-C₄ alkynyl, 6) ←C₃-C₇ cycloalkyl, 7) ←C₃-C₇ cycloalkenyl, 8) ←aryl,9) ←heteroaryl, 10) ←heterocyclyl, 11) ←heterobicyclyl, 12) ←C(O)—R¹¹,13) ←C(O)O—R¹¹, 14) ←C(═Y)NR⁸R⁹, or 15) ←S(O)₂—R¹¹, wherein the alkyl,alkenyl, alkynyl, cycloalkyl, cycloalkenyl is optionally substitutedwith one or more R⁶ substituents; and wherein the aryl, heteroaryl,heterocyclyl, and heterobicyclyl is optionally substituted with one ormore R¹⁰ substituents; R⁶ is 1) halogen, 2) NO₂, 3) CN, 4) haloalkyl, 5)C₁-C₆ alkyl, 6) C₂-C₆ alkenyl, 7) C₂-C₄ alkynyl, 8) C₃-C₇ cycloalkyl, 9)C₃-C₇ cycloalkenyl, 10) aryl, 11) heteroaryl, 12) heterocyclyl, 13)heterobicyclyl, 14) OR ⁷, 15) S(O)_(m)R⁷, 16) NR⁸R⁹, 17) NR⁸S(O)₂R¹¹,18) COR⁷, 19) C(O)OR⁷, 20) CONR⁸R⁹, 21) S(O)₂NR⁸R⁹ 22) OC(O)R⁷, 23)OC(O)Y—R¹¹, 24) SC(O)R⁷, or 25) NC(Y)NR⁸R⁹, wherein the aryl,heteroaryl, heterocyclyl, and heterobicyclyl is optionally substitutedwith one or more R¹⁰ substituents; R⁷ is 1) H, 2) haloalkyl, 3) C₁-C₆alkyl, 4) C₂-C₆ alkenyl, 5) C₂-C₄ alkynyl, 6) C₃-C₇ cycloalkyl, 7) C₃-C₇cycloalkenyl, 8) aryl, 9) heteroaryl, 10) heterocyclyl, 11)heterobicyclyl, 12) R⁸R⁹NC(═Y), or 13) C₁-C₆ alkyl-C₂-C₄ alkenyl, or 14)C₁-C₆ alkyl-C₂-C₄ alkynyl, wherein the alkyl, alkenyl, alkynyl,cycloalkyl, cycloalkenyl is optionally substituted with one or more R⁶substituents; and wherein the aryl, heteroaryl, heterocyclyl, andheterobicyclyl is optionally substituted with one or more R¹⁰substituents; R⁸ and R⁹ are each independently 1) H, 2) haloalkyl, 3)C₁-C₆ alkyl, 4) C₂-C₆ alkenyl, 5) C₂-C₄ alkynyl, 6) C₃-C₇ cycloalkyl, 7)C₃-C₇ cycloalkenyl, 8) aryl, 9) heteroaryl, 10) heterocyclyl, 11)heterobicyclyl, 12) C(O)R¹¹, 13) C(O)Y—R¹¹, or 14) S(O)₂—R¹¹, whereinthe alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl is optionallysubstituted with one or more R⁶ substituents; and wherein the aryl,heteroaryl, heterocyclyl, and heterobicyclyl is optionally substitutedwith one or more R¹⁰ substituents; or R⁸ and R⁹ together with thenitrogen atom to which they are bonded form a five, six or sevenmembered heterocyclic ring optionally substituted with one or more R⁶substituents; R¹⁰ is 1) halogen, 2) NO₂, 3) CN, 4) B(OR¹³)(OR¹⁴), 5)C₁-C₆ alkyl, 6) C₂-C₆ alkenyl, 7) C₂-C₄ alkynyl, 8) C₃-C₇ cycloalkyl, 9)C₃-C₇ cycloalkenyl, 10) haloalkyl, 11) OR⁷, 12) NR⁸R⁹, 13) SR⁷, 14)COR⁷, 15) C(O)OR⁷, 16) S(O)_(m)R⁷, 17) CONR⁸R⁹, 18) S(O)₂NR⁸R⁹, 19)aryl, 20) heteroaryl, 21) heterocyclyl, or 22) heterobicyclyl, whereinthe alkyl, alkenyl, alkynyl, cycloalkyl, and cycloalkenyl is optionallysubstituted with one or more R⁶ substituents; R¹¹ is 1) haloalkyl, 2)C₁-C₆ alkyl, 3) C₂-C₆ alkenyl, 4) C₂-C₄ alkynyl, 5) C₃-C₇ cycloalkyl, 6)C₃-C₇ cycloalkenyl, 7) aryl, 8) heteroaryl, 9) heterocyclyl, or 10)heterobicyclyl, wherein the alkyl, alkenyl, alkynyl, cycloalkyl,cycloalkenyl is optionally substituted with one or more R⁶ substituents;and wherein the aryl, heteroaryl, heterocyclyl, and heterobicyclyl isoptionally substituted with one or more R¹⁰ substituents; R¹² is 1)haloalkyl, 2) C₁-C₆ alkyl, 3) C₂-C₆ alkenyl, 4) C₂-C₄ alkynyl, 5) C₃-C₇cycloalkyl, 6) C₃-C₇ cycloalkenyl, 7) aryl, 8) heteroaryl, 9)heterocyclyl, 10) heterobicyclyl, 11) C(O)—R¹¹, 12) C(O)O—R¹¹, 13)C(O)NR⁸R⁹, 14) S(O)_(m)—R¹¹, or 15) C(═Y)NR⁸R⁹, wherein the alkyl,alkenyl, alkynyl, cycloalkyl, cycloalkenyl is optionally substitutedwith one or more R⁶ substituents; and wherein the aryl, heteroaryl,heterocyclyl, and heterobicyclyl is optionally substituted with one ormore R¹⁰ substituents; R¹³ and R¹⁴ are each independently 1) H, or 2)C₁-C₆ alkyl; or R¹³ and R¹⁴ are combined to form a heterocyclic ring ora heterobicyclyl ring; or a prodrug; or the compound of Formula I islabeled with a detectable label or an affinity tag.
 2. The compound,according to claim 1, in which the compound is a salt.
 3. The compound,according to claim 1, in which the compound is a pharmaceuticallyacceptable salt.
 4. The compound, according to claim 1, in which A andA¹ are both C═O.
 5. The compound, according to claim 1, in which the Aand A¹ are both CH₂.
 6. A compound, according to claim 1, selected fromthe group consisting of: Compound Structure 1

2

3

4

5

6

7

8

9

10

23

24

26

27

28

29


7. A pharmaceutical composition comprising a therapeutically effectiveamount of a compound, according to claim 1, and a pharmaceuticallyacceptable carrier.
 8. A method for the treatment or prevention of aproliferative disorder in a subject, the method comprising:administering to the subject a therapeutically effective amount of thecompound, according to claim
 1. 9. The method, according to claim 8, inwhich the proliferative disorder is cancer.
 10. The method, according toclaim 8, further comprising administering to the subject atherapeutically effective amount of a chemotherapeutic agent prior to,simultaneously with or after administration of the compound.
 11. Themethod, according to claim 8, further comprising administering to thesubject a therapeutically effective amount of a death receptor agonistprior to, simultaneously with or after administration of the compound.12. The method, according to claim 11, in which the death receptoragonist is TRAIL.
 13. The method, according to claim 11, in which thedeath receptor agonist is a TRAIL antibody.
 14. The method, according toclaim 11, in which the death receptor agonist is administered in anamount that produces a synergistic effect.